3,775 results on '"Kinase activity"'
Search Results
2. Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity.
- Author
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Pogacar Z, Johnson JL, Krenning L, De Conti G, Jochems F, Lieftink C, Velds A, Wardak L, Groot K, Schepers A, Wang L, Song JY, van de Ven M, van Tellingen O, Medema RH, Beijersbergen RL, Bernards R, and Leite de Oliveira R
- Subjects
- Cell Line, Tumor, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinase 4 metabolism, Humans, Piperazines, Pyridines, Sulfonamides, Cyclin-Dependent Kinase 6 metabolism, Protein Kinase Inhibitors pharmacology
- Abstract
Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition., Competing Interests: R.B is the founder of the company Oncosence (https://www.oncosence.com), which aims to develop senescence-inducing and senolytic compounds to treat cancer. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
- Published
- 2022
- Full Text
- View/download PDF
3. pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex
- Author
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Bruce W. Banfield, Michael A. Gulak, Jamil H. Muradov, Thomas J. M. Hay, and Renée L. Finnen
- Subjects
Physiology ,viruses ,Herpesvirus 2, Human ,Cultured tumor cells ,medicine.disease_cause ,Pathology and Laboratory Medicine ,Biochemistry ,Chlorocebus aethiops ,Medicine and Health Sciences ,Biology (General) ,Post-Translational Modification ,Phosphorylation ,Virus Release ,0303 health sciences ,Chemistry ,Kinase ,030302 biochemistry & molecular biology ,Viral tegument ,Transfection ,3. Good health ,Cell biology ,Body Fluids ,medicine.anatomical_structure ,Blood ,Herpes Simplex Virus-2 ,Medical Microbiology ,Viral Pathogens ,Viruses ,Cell lines ,Cellular Structures and Organelles ,Anatomy ,Pathogens ,Biological cultures ,Research Article ,Herpesviruses ,QH301-705.5 ,Viral protein ,Imaging Techniques ,Nuclear Envelope ,Immunology ,Phosphatase ,DNA construction ,Protein Serine-Threonine Kinases ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Viral Proteins ,Capsid ,Nuclear Membrane ,Virology ,Fluorescence Imaging ,Genetics ,medicine ,Inner membrane ,Animals ,Humans ,HeLa cells ,Nuclear membrane ,Kinase activity ,Molecular Biology Techniques ,Molecular Biology ,Microbial Pathogens ,Vero Cells ,030304 developmental biology ,Cell Nucleus ,Virus Assembly ,Organisms ,Biology and Life Sciences ,Proteins ,Herpes Simplex ,Cell Biology ,RC581-607 ,Blood Serum ,Cell cultures ,digestive system diseases ,Herpes Simplex Virus ,Herpes simplex virus ,Cytoplasm ,Plasmid Construction ,Parasitology ,Immunologic diseases. Allergy ,DNA viruses ,Immune Serum - Abstract
It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope., Author summary During all herpesvirus infections, the nuclear envelope undergoes deformation in order to enable viral capsids assembled within the nucleus of the infected cell to gain access to the cytoplasm for further maturation and spread to neighbouring cells. These nuclear envelope deformations are orchestrated by the viral nuclear egress complex (NEC), which, in HSV, is composed of two viral proteins, pUL31 and pUL34. How the membrane-deformation activity of the NEC is controlled during infection is incompletely understood. The studies in this communication reveal that the phosphorylation status of pUL31 and pUL34 can determine the nature of nuclear envelope deformations and that the viral protein pUL21 can modulate the phosphorylation status of both NEC components. These findings provide an explanation for why HSV-2 strains lacking pUL21 are defective in nuclear egress. A thorough understanding of how NEC activity is controlled during infection may yield strategies to disrupt this fundamental step in the herpesvirus lifecycle, providing the basis for novel antiviral strategies.
- Published
- 2021
4. Structure and kinase activity of bacterial cell cycle regulator CcrZ.
- Author
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Wozniak KJ, Burby PE, Nandakumar J, and Simmons LA
- Subjects
- Bacillus subtilis genetics, Bacillus subtilis metabolism, Bacterial Proteins metabolism, Cell Cycle genetics, Choline metabolism, DNA Replication genetics, DNA-Binding Proteins genetics, Ribose metabolism
- Abstract
CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
- Full Text
- View/download PDF
5. The SH2 domain and kinase activity of JAK2 target JAK2 to centrosome and regulate cell growth and centrosome amplification.
- Author
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Shahi, Aashirwad, Kahle, Jacob, Hopkins, Chandler, and Diakonova, Maria
- Subjects
- *
CELL growth , *CELLULAR control mechanisms , *PROTEIN-tyrosine kinases , *CELL cycle , *REGULATION of growth - Abstract
JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S −> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
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6. pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex.
- Author
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Muradov JH, Finnen RL, Gulak MA, Hay TJM, and Banfield BW
- Subjects
- Animals, Capsid physiology, Cell Nucleus genetics, Cell Nucleus metabolism, Chlorocebus aethiops, HeLa Cells, Herpes Simplex metabolism, Herpes Simplex virology, Humans, Nuclear Envelope metabolism, Nuclear Envelope virology, Phosphorylation, Protein Serine-Threonine Kinases genetics, Vero Cells, Viral Proteins genetics, Virus Assembly, Herpes Simplex pathology, Herpesvirus 2, Human physiology, Nuclear Envelope pathology, Protein Serine-Threonine Kinases metabolism, Viral Proteins metabolism, Virus Release
- Abstract
It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
- View/download PDF
7. Ca2+/calmodulin and apo-calmodulin both bind to and enhance the tyrosine kinase activity of c-Src
- Author
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Fondo Nacional de Ciencia, Tecnología e Innovación (Venezuela), Universidad Central de Venezuela, Consejo de Desarrollo Científico, Humanístico, Tecnológico y de las Artes (Venezuela), European Commission, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Consejo Superior de Investigaciones Científicas (España), Stateva, Silvia R., Salas, Valentina, Anguita, Estefanía, Benaim, Gustavo, Villalobo, Antonio, Fondo Nacional de Ciencia, Tecnología e Innovación (Venezuela), Universidad Central de Venezuela, Consejo de Desarrollo Científico, Humanístico, Tecnológico y de las Artes (Venezuela), European Commission, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Consejo Superior de Investigaciones Científicas (España), Stateva, Silvia R., Salas, Valentina, Anguita, Estefanía, Benaim, Gustavo, and Villalobo, Antonio
- Abstract
Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2 +-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.
- Published
- 2015
8. Autophosphorylation of Ser-6 via an intermolecular mechanism is important for the rapid reduction of NtCDPK1 kinase activity for substrate RSG
- Author
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Takeshi Ito, Sarahmi Ishida, and Yohsuke Takahashi
- Subjects
0301 basic medicine ,Nicotiana tabacum ,Cell Membranes ,lcsh:Medicine ,Plant Science ,Biochemistry ,Physical Chemistry ,Catalytic Domain ,Serine ,Homeostasis ,Post-Translational Modification ,Phosphorylation ,Plant Hormones ,lcsh:Science ,Plant Proteins ,Multidisciplinary ,biology ,Chemistry ,Plant Biochemistry ,Autophosphorylation ,Plants, Genetically Modified ,Glutathione ,Cell biology ,Enzymes ,Basic-Leucine Zipper Transcription Factors ,Plant Physiology ,Physical Sciences ,Plant hormone ,Cellular Structures and Organelles ,Dimerization ,Research Article ,Immunoblotting ,Molecular Probe Techniques ,Research and Analysis Methods ,03 medical and health sciences ,Tobacco ,Kinase activity ,Protein kinase A ,Molecular Biology Techniques ,Psychological repression ,Transcription factor ,Molecular Biology ,lcsh:R ,Biology and Life Sciences ,Proteins ,Membrane Proteins ,Cell Biology ,biology.organism_classification ,Hormones ,Gibberellins ,Enzyme Activation ,Repressor Proteins ,030104 developmental biology ,Chemical Properties ,Enzymology ,lcsh:Q ,Peptides ,Protein Kinases ,Protein Processing, Post-Translational - Abstract
Tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase 1 (NtCDPK1) is involved in feedback regulation of the plant hormone gibberellin through the phosphorylation of the transcription factor, REPRESSION OF SHOOT GROWTH (RSG). Previously, Ser-6 and Thr-21 were identified as autophosphorylation sites in NtCDPK1. Autophosphorylation of Ser-6 and Thr-21 not only decreases the binding affinity of NtCDPK1 for RSG, but also inhibits the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreases the phosphorylation efficiency of RSG. We demonstrated that Ser-6 and Thr-21 of NtCDPK1 are phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhances the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These findings suggested that autophosphorylation of Ser-6 and Thr-21 prevents excessive phosphorylation of RSG. In this study, we attempted to determine which autophosphorylation site is responsible for the functional regulation of NtCDPK1. Ser-6 was autophosphorylated within 1 min, whereas Thr-21 required over 5 min to be completely autophosphorylated. Furthermore, we found that Ser-6 and Thr-21 were autophosphorylated by inter- and intramolecular mechanisms, respectively, which may be reflected in the faster autophosphorylation of Ser-6. Although both autophosphorylation sites were involved in the reduction of the binding affinity of NtCDPK1 for RSG and the inhibition of NtCDPK1 homodimerization, autophosphorylation of Ser-6 alone was sufficient to decrease the kinase activity of NtCDPK1 for RSG. These results suggest that autophosphorylation of Ser-6 is important for the rapid reduction of NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role.
- Published
- 2018
9. ADP is the dominant controller of AMP-activated protein kinase activity dynamics in skeletal muscle during exercise.
- Author
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Coccimiglio IF and Clarke DC
- Subjects
- Animals, Computational Biology, Humans, Mice, Models, Biological, Signal Transduction physiology, AMP-Activated Protein Kinases chemistry, AMP-Activated Protein Kinases metabolism, Adenosine Diphosphate chemistry, Adenosine Diphosphate metabolism, Adenosine Diphosphate pharmacokinetics, Exercise physiology, Muscle, Skeletal enzymology, Muscle, Skeletal metabolism, Muscle, Skeletal physiology
- Abstract
Exercise training elicits profound metabolic adaptations in skeletal muscle cells. A key molecule in coordinating these adaptations is AMP-activated protein kinase (AMPK), whose activity increases in response to cellular energy demand. AMPK activity dynamics are primarily controlled by the adenine nucleotides ADP and AMP, but how each contributes to its control in skeletal muscle during exercise is unclear. We developed and validated a mathematical model of AMPK signaling dynamics, and then applied global parameter sensitivity analyses with data-informed constraints to predict that AMPK activity dynamics are determined principally by ADP and not AMP. We then used the model to predict the effects of two additional direct-binding activators of AMPK, ZMP and Compound 991, further validating the model and demonstrating its applicability to understanding AMPK pharmacology. The relative effects of direct-binding activators can be understood in terms of four properties, namely their concentrations, binding affinities for AMPK, abilities to enhance AMPK phosphorylation, and the magnitudes of their allosteric activation of AMPK. Despite AMP's favorable values in three of these four properties, ADP is the dominant controller of AMPK activity dynamics in skeletal muscle during exercise by virtue of its higher concentration compared to that of AMP., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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10. The CaMKII K42M and K42R mutations are equivalent in suppressing kinase activity and targeting.
- Author
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Tullis JE, Rumian NL, Brown CN, and Bayer KU
- Subjects
- Animals, Calcium metabolism, Calcium-Calmodulin-Dependent Protein Kinase Type 2 chemistry, Cells, Cultured, Female, Glutamic Acid pharmacology, HEK293 Cells, Hippocampus cytology, Humans, Male, Movement, Phosphorylation, Rats, Sprague-Dawley, Receptors, N-Methyl-D-Aspartate metabolism, Synapses metabolism, Calcium-Calmodulin-Dependent Protein Kinase Type 2 genetics, Mutation genetics
- Abstract
CaMKII is an important mediator of forms of synaptic plasticity that are thought to underly learning and memory. The CaMKII mutants K42M and K42R have been used interchangeably as research tools, although some reported phenotypic differences suggest that they may differ in the extent to which they impair ATP binding. Here, we directly compared the two mutations at the high ATP concentrations that exist within cells (~4 mM). We found that both mutations equally blocked GluA1 phosphorylation in vitro and GluN2B binding within cells. Both mutations also reduced but did not completely abolish CaMKII T286 autophosphorylation in vitro or CaMKII movement to excitatory synapses in neurons. Thus, despite previously suggested differences, both mutations appear to interfere with ATP binding to the same extent., Competing Interests: No authors have competing interests.
- Published
- 2020
- Full Text
- View/download PDF
11. Correction: S-Nitrosylation of G protein-coupled receptor kinase 6 and Casein kinase 2 alpha modulates their kinase activity toward alpha-synuclein phosphorylation in an animal model of Parkinson's disease.
- Author
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Wu W, Sung CC, Yu P, Li J, and Chung KKK
- Abstract
[This corrects the article DOI: 10.1371/journal.pone.0232019.].
- Published
- 2020
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12. S-Nitrosylation of G protein-coupled receptor kinase 6 and Casein kinase 2 alpha modulates their kinase activity toward alpha-synuclein phosphorylation in an animal model of Parkinson's disease.
- Author
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Wu W, Sung CC, Yu P, Li J, and Chung KKK
- Subjects
- Age Factors, Animals, Casein Kinase II chemistry, Disease Models, Animal, G-Protein-Coupled Receptor Kinases chemistry, Gene Deletion, HEK293 Cells, Humans, Mice, Mice, Transgenic, Mutation, Nitric Oxide Synthase Type I genetics, Nitroarginine administration & dosage, Nitroarginine pharmacology, Nitrosative Stress, Parkinson Disease drug therapy, Parkinson Disease genetics, Phosphorylation, Serine metabolism, alpha-Synuclein chemistry, Casein Kinase II metabolism, G-Protein-Coupled Receptor Kinases metabolism, Nitric Oxide metabolism, Parkinson Disease metabolism, alpha-Synuclein genetics, alpha-Synuclein metabolism
- Abstract
Parkinson's disease (PD) is a common neurodegenerative disorder which is mostly sporadic but familial-linked PD (FPD) cases have also been found. The first reported gene mutation that linked to PD is α-synuclein (α-syn). Studies have shown that mutations, increased expression or abnormal processing of α-syn can contribute to PD, but it is believed that multiple mechanisms are involved. One of the contributing factors is post-translational modification (PTM), such as phosphorylation of α-syn at serine 129 by G-protein-coupled receptor kinases (GRKs) and casein kinase 2α (CK2α). Another known important contributing factor to PD pathogenesis is oxidative and nitrosative stress. In this study, we found that GRK6 and CK2α can be S-nitrosylated by nitric oxide (NO) both in vitro and in vivo. S-nitrosylation of GRK6 and CK2α enhanced their kinase activity towards the phosphorylation of α-syn at S129. In an A53T α-syn transgenic mouse model of PD, we found that increased GRK6 and CK2α S-nitrosylation were observed in an age dependent manner and it was associated with an increased level of pSer129 α-syn. Treatment of A53T α-syn transgenic mice with Nω-Nitro-L-arginine (L-NNA) significantly reduced the S-nitrosylation of GRK6 and CK2α in the brain. Finally, deletion of neuronal nitric oxide synthase (nNOS) in A53T α-syn transgenic mice reduced the levels of pSer129 α-syn and α-syn in an age dependent manner. Our results provide a novel mechanism of how NO through S-nitrosylation of GRK6 and CK2α can enhance the phosphorylation of pSer129 α-syn in an animal model of PD., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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13. The KLDpT activation loop motif is critical for MARK kinase activity.
- Author
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Sonntag T, Moresco JJ, Yates JR 3rd, and Montminy M
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Cell Membrane metabolism, Conserved Sequence, Cyclic AMP metabolism, HEK293 Cells, Humans, Mice, Microtubules metabolism, Models, Molecular, Phosphorylation, Protein Binding, Protein Domains, Protein Structure, Secondary, Structure-Activity Relationship, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases metabolism
- Abstract
MAP/microtubule-affinity regulating kinases (MARK1-4) are members of the AMPK family of Ser/Thr-specific kinases, which phosphorylate substrates at consensus LXRXXSXXXL motifs. Within microtubule-associated proteins, MARKs also mediate phosphorylation of variant KXGS or ζXKXGSXXNΨ motifs, interfering with the ability of tau and MAP2/4 to bind to microtubules. Here we show that, although MARKs and the closely related salt-inducible kinases (SIKs) phosphorylate substrates with consensus AMPK motifs comparably, MARKs are more potent in recognizing variant ζXKXGSXXNΨ motifs on cellular tau. In studies to identify regions of MARKs that confer catalytic activity towards variant sites, we found that the C-terminal kinase associated-1 (KA1) domain in MARK1-3 mediates binding to microtubule-associated proteins CLASP1/2; but this interaction is dispensable for ζXKXGSXXNΨ phosphorylation. Mutational analysis of MARK2 revealed that the N-terminal kinase domain of MARK2 is sufficient for phosphorylation of both consensus and variant ζXKXGSXXNΨ sites. Within this domain, the KLDpT activation loop motif promotes MARK2 activity both intracellularly and in vitro, but has no effect on SIK2 activity. As KLDpT is conserved in all vertebrates MARKs, we conclude that this sequence is crucial for MARK-dependent regulation of cellular polarity., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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14. Ethyl pyruvate emerges as a safe and fast acting agent against Trypanosoma brucei by targeting pyruvate kinase activity
- Author
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Universität Leipzig, Missionsärztliches Institut Würzburg, Universitätsklinikum Leipzig, Worku, Netsanet, Stich, August, Daugschies, Arwid, Wenzel, Iris, Kurz, Randy, Thieme, Rene, Kurz, Susanne, Birkenmeier, Gerd, Universität Leipzig, Missionsärztliches Institut Würzburg, Universitätsklinikum Leipzig, Worku, Netsanet, Stich, August, Daugschies, Arwid, Wenzel, Iris, Kurz, Randy, Thieme, Rene, Kurz, Susanne, and Birkenmeier, Gerd
- Abstract
Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. Results: The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. Conclusion: Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability t
- Published
- 2015
15. Critical role of kinase activity of hematopoietic progenitor kinase 1 in anti-tumor immune surveillance.
- Author
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Liu, Jinqi, Curtin, Joshua, You, Dan, Hillerman, Stephen, Li-Wang, Bifang, Eraslan, Rukiye, Xie, Jenny, Swanson, Jesse, Ho, Ching-Ping, Oppenheimer, Simone, Warrack, Bethanne M., McNaney, Colleen A., Nelson, David M., Blum, Jordan, Kim, Taeg, Fereshteh, Mark, Reily, Michael, Shipkova, Petia, Murtaza, Anwar, and Sanjuan, Miguel
- Subjects
- *
CYTOTOXIC T cells , *T cells , *NATURAL immunity , *IMMUNE response , *DENDRITIC cells , *DRUG resistance in cancer cells - Abstract
Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of—function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
16. Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity
- Author
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Jie Ming, Bo Tian, Pei Zhang, Yang Shu, Qingzhi Wang, and Fengjuan Jiao
- Subjects
0301 basic medicine ,1-Methyl-4-phenylpyridinium ,lcsh:Medicine ,Mitogen-activated protein kinase kinase ,Toxicology ,Pathology and Laboratory Medicine ,Biochemistry ,MAP2K7 ,0302 clinical medicine ,Animal Cells ,Medicine and Health Sciences ,Serine ,ASK1 ,Post-Translational Modification ,Phosphorylation ,lcsh:Science ,Cells, Cultured ,Neurons ,Mice, Knockout ,Neuronal Death ,Multidisciplinary ,Movement Disorders ,biology ,Cell Death ,In Vitro Kinase Assay ,Neurodegenerative Diseases ,Parkinson Disease ,Precipitation Techniques ,Up-Regulation ,Bioassays and Physiological Analysis ,Neurology ,Cell Processes ,Cellular Types ,Research Article ,Protein Binding ,Immunoblotting ,Molecular Sequence Data ,Mutation, Missense ,Mice, Transgenic ,Protein Serine-Threonine Kinases ,Research and Analysis Methods ,Transfection ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,03 medical and health sciences ,Immunoprecipitation ,Animals ,Humans ,Amino Acid Sequence ,Kinase activity ,Molecular Biology Techniques ,Molecular Biology ,Enzyme Assays ,MAP kinase kinase kinase ,Toxicity ,Herbicides ,Cyclin-dependent kinase 5 ,Cyclin-dependent kinase 2 ,lcsh:R ,Biology and Life Sciences ,Proteins ,Cyclin-Dependent Kinase 5 ,Cell Biology ,Molecular biology ,nervous system diseases ,030104 developmental biology ,HEK293 Cells ,nervous system ,biology.protein ,Cyclin-dependent kinase 9 ,lcsh:Q ,Biochemical Analysis ,030217 neurology & neurosurgery - Abstract
Background Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown. Principle Findings Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner. Conclusion Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.
- Published
- 2016
17. Ca2+/calmodulin and apo-calmodulin both bind to and enhance the tyrosine kinase activity of c-Src
- Author
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Silviya R. Stateva, Estefanía Anguita, Gustavo Benaim, Valentina Salas, Antonio Villalobo, Fondo Nacional de Ciencia, Tecnología e Innovación (Venezuela), Universidad Central de Venezuela, Consejo de Desarrollo Científico, Humanístico, Tecnológico y de las Artes (Venezuela), European Commission, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), and Consejo Superior de Investigaciones Científicas (España)
- Subjects
Calmodulin ,lcsh:Medicine ,ComputingMilieux_LEGALASPECTSOFCOMPUTING ,Biology ,SH3 domain ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Humans ,Kinase activity ,lcsh:Science ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Tyrosine-protein kinase CSK ,Kinase ,lcsh:R ,Molecular biology ,3. Good health ,Cell biology ,src-Family Kinases ,biology.protein ,Calcium ,lcsh:Q ,Tyrosine kinase ,A431 cells ,030217 neurology & neurosurgery ,Research Article ,Protein Binding ,Proto-oncogene tyrosine-protein kinase Src - Abstract
This is an open access article distributed under the terms of the Creative Commons Attribution License., Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2 +-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred., This work was funded by grants to AV from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494 & SAF2014-52048-R), the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349), the CSIC program iCOOP+ 2014 (COOPA20053), and the European Commission (contract PITN-GA-2011-289033). SRS received funding from the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program FP7/2007-2013 under REA grant agreement n° PITN-GA-2011-289033. VS and GB were supported by fellowship and grants from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (03-00-6057-2005 & PG-03-8728-2013) and Fondo Nacional de Ciencia, Tecnología e Innovación (P-2011000884)
- Published
- 2015
18. Autophosphorylation of Ser-6 via an intermolecular mechanism is important for the rapid reduction of NtCDPK1 kinase activity for substrate RSG.
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Ito, Takeshi, Ishida, Sarahmi, and Takahashi, Yohsuke
- Subjects
- *
AUTOPHOSPHORYLATION , *KINASES , *GIBBERELLINS , *TOBACCO , *TRANSCRIPTION factors , *PHYSIOLOGY - Abstract
Tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase 1 (NtCDPK1) is involved in feedback regulation of the plant hormone gibberellin through the phosphorylation of the transcription factor, REPRESSION OF SHOOT GROWTH (RSG). Previously, Ser-6 and Thr-21 were identified as autophosphorylation sites in NtCDPK1. Autophosphorylation of Ser-6 and Thr-21 not only decreases the binding affinity of NtCDPK1 for RSG, but also inhibits the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreases the phosphorylation efficiency of RSG. We demonstrated that Ser-6 and Thr-21 of NtCDPK1 are phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhances the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These findings suggested that autophosphorylation of Ser-6 and Thr-21 prevents excessive phosphorylation of RSG. In this study, we attempted to determine which autophosphorylation site is responsible for the functional regulation of NtCDPK1. Ser-6 was autophosphorylated within 1 min, whereas Thr-21 required over 5 min to be completely autophosphorylated. Furthermore, we found that Ser-6 and Thr-21 were autophosphorylated by inter- and intramolecular mechanisms, respectively, which may be reflected in the faster autophosphorylation of Ser-6. Although both autophosphorylation sites were involved in the reduction of the binding affinity of NtCDPK1 for RSG and the inhibition of NtCDPK1 homodimerization, autophosphorylation of Ser-6 alone was sufficient to decrease the kinase activity of NtCDPK1 for RSG. These results suggest that autophosphorylation of Ser-6 is important for the rapid reduction of NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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19. SpdC, a novel virulence factor, controls histidine kinase activity in Staphylococcus aureus.
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Poupel O, Proux C, Jagla B, Msadek T, and Dubrac S
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- Amino Acid Sequence, Animals, Bacterial Proteins genetics, Biofilms growth & development, Female, Histidine Kinase genetics, Mice, Phosphorylation, Regulon, Sepsis metabolism, Signal Transduction, Staphylococcal Infections metabolism, Staphylococcus aureus pathogenicity, Virulence, Virulence Factors genetics, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial, Histidine Kinase metabolism, Sepsis microbiology, Staphylococcal Infections microbiology, Virulence Factors metabolism
- Abstract
The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons.
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- 2018
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20. Pharmacologic Inhibition of MLK3 Kinase Activity Blocks the In Vitro Migratory Capacity of Breast Cancer Cells but Has No Effect on Breast Cancer Brain Metastasis in a Mouse Xenograft Model
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Kun Hyoe Rhoo, Joynita Sur, Changyong Feng, Harris A. Gelbard, Megan Granger, Stephen Dewhurst, and Oksana Polesskaya
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CA15-3 ,Pathology ,Pyridines ,Cell ,lcsh:Medicine ,Metastasis ,Mice ,0302 clinical medicine ,Cell Signaling ,Cell Movement ,Breast Tumors ,Medicine and Health Sciences ,Medicine ,lcsh:Science ,skin and connective tissue diseases ,Neurological Tumors ,0303 health sciences ,Multidisciplinary ,Brain Neoplasms ,MAP Kinase Kinase Kinases ,Cell Motility ,medicine.anatomical_structure ,Neurology ,Oncology ,030220 oncology & carcinogenesis ,Female ,Research Article ,Signal Transduction ,medicine.medical_specialty ,Transplantation, Heterologous ,CA 15-3 ,Mice, Nude ,Breast Neoplasms ,Cell Migration ,03 medical and health sciences ,Breast cancer ,Cell Line, Tumor ,Breast Cancer ,Animals ,Pyrroles ,Kinase activity ,Protein Kinase Inhibitors ,030304 developmental biology ,business.industry ,Cell growth ,lcsh:R ,Biology and Life Sciences ,Cancers and Neoplasms ,Cell Biology ,medicine.disease ,Disease Models, Animal ,Cancer research ,Brain Metastasis ,lcsh:Q ,business ,Neoplasm Transplantation ,Brain metastasis - Abstract
Brain metastasis of breast cancer is an important clinical problem, with few therapeutic options and a poor prognosis. Recent data have implicated mixed lineage kinase 3 (MLK3) in controlling the in vitro migratory capacity of breast cancer cells, as well as the metastasis of MDA-MB-231 breast cancer cells from the mammary fat pad to distant lymph nodes in a mouse xenograft model. We therefore set out to test whether MLK3 plays a role in brain metastasis of breast cancer cells. To address this question, we used a novel, brain penetrant, MLK3 inhibitor, URMC099. URMC099 efficiently inhibited the migration of breast cancer cells in an in vitro cell monolayer wounding assay, and an in vitro transwell migration assay, but had no effect on in vitro cell growth. We also tested the effect of URMC099 on tumor formation in a mouse xenograft model of breast cancer brain metastasis. This analysis showed that URMC099 had no effect on the either the frequency or size of breast cancer brain metastases. We conclude that pharmacologic inhibition of MLK3 by URMC099 can reduce the in vitro migratory capacity of breast cancer cells, but that it has no effect on either the frequency or size of breast cancer brain metastases, in a mouse xenograft model.
- Published
- 2014
21. Cardiac-Specific Inhibition of Kinase Activity in Calcium/Calmodulin-Dependent Protein Kinase Kinase-β Leads to Accelerated Left Ventricular Remodeling and Heart Failure after Transverse Aortic Constriction in Mice
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Takeshi Kimura, Shin Watanabe, Michiko Narazaki, Osamu Baba, Takahiro Horie, Genzou Takemura, Koji Hasegawa, Hitoo Nishi, Koh Ono, Hiromichi Wada, Tetsuya Matsuda, Naoya Sowa, Yasuhide Kuwabara, and Kazuya Nagao
- Subjects
Male ,medicine.medical_specialty ,Magnetic Resonance Spectroscopy ,Calmodulin ,Heart Ventricles ,Cardiac Hypertrophy ,Cardiology ,lcsh:Medicine ,Calcium-Calmodulin-Dependent Protein Kinase Kinase ,Mice, Transgenic ,Mitochondria, Heart ,Mice ,Ventricular Dysfunction, Left ,Adenosine Triphosphate ,Internal medicine ,medicine ,Medicine and Health Sciences ,Animals ,Kinase activity ,Phosphorylation ,Protein kinase A ,Ventricular remodeling ,lcsh:Science ,Promoter Regions, Genetic ,Molecular Biology ,Pressure overload ,Heart Failure ,Multidisciplinary ,biology ,Myosin Heavy Chains ,Ventricular Remodeling ,Kinase ,Chemistry ,lcsh:R ,AMPK ,Biology and Life Sciences ,medicine.disease ,Up-Regulation ,Disease Models, Animal ,Endocrinology ,Gene Expression Regulation ,biology.protein ,lcsh:Q ,Signal transduction ,Research Article ,Signal Transduction - Abstract
[Background]The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated. [Methods and Results]We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) transgenic (α-MHC CaMKKβ[kd] TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKβ activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKβ[kd] TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKβ downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKβ[kd] TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKβ and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKβ[kd] TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/β-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKβ[kd] TG mice compared with WT mice after TAC. [Conclusions]These data indicate that CaMKKβ exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKβ may be a therapeutic target for the treatment of heart failure.
- Published
- 2014
22. mTOR has a developmental stage-specific role in mitochondrial fitness independent of conventional mTORC1 and mTORC2 and the kinase activity.
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Kalim, Khalid W., Zhang, Shuangmin, Chen, Xiaoyi, Li, Yuan, Yang, Jun-Qi, Zheng, Yi, and Guo, Fukun
- Subjects
- *
MAMMALS , *RAPAMYCIN , *MITOCHONDRIAL DNA , *PROTEINS , *GROWTH factors - Abstract
The mammalian target of rapamycin (mTOR), present in mTOR complex 1 (mTORC1) and mTORC2, is a serine/threonine kinase that integrates nutrients, growth factors, and cellular energy status to control protein synthesis, cell growth, survival and metabolism. However, it remains elusive whether mTOR plays a developmental stage-specific role in tissue development and whether mTOR can function independent of its complexes and kinase activity. In this study, by inducible genetic manipulation approach, we investigated the role of mTOR and its dependence on mTOR complexes and kinase activity in mitochondrial fitness of early, progenitor stage (lineage-negative; Lin-) versus later, lineage-committed stage (lineage-positive; Lin+) of hematopoietic cells. We found that oxidative phosphorylation (OXPHOS), ATP production and mitochondrial DNA synthesis were decreased in mTOR-/- Lin- cells but increased in mTOR-/- Lin+ cells, suggesting that mTOR plays a developmental stage-specific role in OXPHOS, ATP production and mitochondrial DNA synthesis. In contrast to mTOR deletion, simultaneous deletion of Raptor, a key component of mTORC1, and Rictor, a key component of mTORC2, led to increased mitochondrial DNA in Lin- cells and decreased mitochondrial DNA and ATP production in Lin+ cells, suggesting that mTOR regulates mitochondrial DNA synthesis in Lin- and Lin+ cells and ATP production in Lin+ cells independent of mTORC1 and mTORC2. Similar to mTOR deletion, deletion of Raptor alone attenuated glycolysis and increased mitochondrial mass and mitochondrial membrane potential in Lin- cells and increased mitochondrial mass and OXPHOS in Lin+ cells, whereas deletion of Rictor alone had no effect on these mitochondrial parameters in Lin- and Lin+ cells, suggesting that mTOR regulates glycolysis and mitochondrial membrane potential in Lin- cells, OXPHOS in Lin+ cells, and mitochondrial mass in both Lin- and Lin+ cells dependent on mTORC1, but not mTORC2. Either Raptor deficiency or Rictor deficiency recapitulated mTOR deletion in decreasing OXPHOS in Lin- cells and glycolysis in Lin+ cells, suggesting that mTOR regulates OXPHOS in Lin- cells and glycolysis in Lin+ cells dependent on both mTORC1 and mTORC2. Finally, mice harboring a mTOR kinase dead D2338A knock-in mutant showed decreased glycolysis in Lin+ cells, as seen in mTOR-/- Lin+ cells, but no change in glycolysis in Lin- cells, in contrast to the decreased glycolysis in mTOR-/- Lin- cells, suggesting that mTOR regulates glycolysis in Lin+ cells dependent on its kinase activity, whereas mTOR regulates glycolysis in Lin- cells independent of its kinase activity. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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23. The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation.
- Author
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Guerrero-Mendiola, Carlos, García-Trejo, José J., Encalada, Rusely, Saavedra, Emma, and Ramírez-Silva, Leticia
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- *
PYRUVATE kinase , *VIBRIO cholerae , *ALLOSTERIC regulation , *ENZYME kinetics , *RIBOSE phosphates - Abstract
In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
24. Increasing creatine kinase activity protects against hypoxia / reoxygenation injury but not against anthracycline toxicity in vitro.
- Author
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Zervou S, Whittington HJ, Ostrowski PJ, Cao F, Tyler J, Lake HA, Neubauer S, and Lygate CA
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- Animals, Base Sequence, Cell Death drug effects, Cell Hypoxia drug effects, Cloning, Molecular, Doxorubicin pharmacology, HEK293 Cells, Humans, Isoenzymes metabolism, Mice, Open Reading Frames genetics, Transfection, Anthracyclines toxicity, Creatine Kinase metabolism, Cytoprotection drug effects, Oxygen toxicity
- Abstract
The creatine kinase (CK) phosphagen system is fundamental to cellular energy homeostasis. Cardiomyocytes express three CK isoforms, namely the mitochondrial sarcomeric CKMT2 and the cytoplasmic CKM and CKB. We hypothesized that augmenting CK in vitro would preserve cell viability and function and sought to determine efficacy of the various isoforms. The open reading frame of each isoform was cloned into pcDNA3.1, followed by transfection and stable selection in human embryonic kidney cells (HEK293). CKMT2- CKM- and CKB-HEK293 cells had increased protein and total CK activity compared to non-transfected cells. Overexpressing any of the three CK isoforms reduced cell death in response to 18h hypoxia at 1% O2 followed by 2h re-oxygenation as assayed using propidium iodide: by 33% in CKMT2, 47% in CKM and 58% in CKB compared to non-transfected cells (P<0.05). Loading cells with creatine did not modify cell survival. Transient expression of CK isoforms in HL-1 cardiac cells elevated isoenzyme activity, but only CKMT2 over-expression protected against hypoxia (0.1% for 24h) and reoxygenation demonstrating 25% less cell death compared to non-transfected control (P<0.01). The same cells were not protected from doxorubicin toxicity (250nM for 48h), in contrast to the positive control. These findings support increased CK activity as protection against ischaemia-reperfusion injury, in particular, protection via CKMT2 in a cardiac-relevant cell line, which merits further investigation in vivo.
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- 2017
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25. The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation.
- Author
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Guerrero-Mendiola C, García-Trejo JJ, Encalada R, Saavedra E, and Ramírez-Silva L
- Subjects
- Allosteric Regulation, Amino Acid Sequence genetics, Animals, Binding Sites, Fructosediphosphates metabolism, Isoenzymes genetics, Kinetics, Pyruvate Kinase genetics, Rabbits, Ribosemonophosphates metabolism, Substrate Specificity, Vibrio cholerae genetics, Isoenzymes metabolism, Potassium metabolism, Pyruvate Kinase metabolism, Vibrio cholerae enzymology
- Abstract
In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may "mimic" the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium.
- Published
- 2017
- Full Text
- View/download PDF
26. Increased nucleoside diphosphate kinase activity induces white spot syndrome virus infection in Litopenaeus vannamei.
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Liu PF, Liu QH, Wu Y, and Huang J
- Subjects
- Animals, Enzyme Activation, Gene Dosage, Gene Expression, Nucleoside-Diphosphate Kinase genetics, Organ Specificity genetics, Penaeidae genetics, RNA Interference, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Nucleoside-Diphosphate Kinase metabolism, Penaeidae enzymology, Penaeidae virology, White spot syndrome virus 1
- Abstract
Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP's) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp.
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- 2017
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27. P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig.
- Author
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Oqani, Reza K., Lin, Tao, Lee, Jae Eun, Choi, Ki Myung, Shin, Hyun Young, and Jin, Dong Il
- Subjects
- *
GENETIC transcription , *ELONGATION factors (Biochemistry) , *RNA polymerase II , *MEIOSIS , *GENETIC regulation , *IMMUNOCYTOCHEMISTRY , *SWINE genetics - Abstract
Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig. [ABSTRACT FROM AUTHOR]
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- 2016
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28. Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity.
- Author
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Shu, Yang, Ming, Jie, Zhang, Pei, Wang, Qingzhi, Jiao, Fengjuan, and Tian, Bo
- Subjects
- *
PARKINSON'S disease , *GENETIC mutation , *PHOSPHORYLATION , *GENETIC regulation , *CAUSES of death - Abstract
Background: Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown. Principle Findings: Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner. Conclusion: Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death. [ABSTRACT FROM AUTHOR]
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- 2016
- Full Text
- View/download PDF
29. A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.
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Ouellette, Steven B., Noel, Brett M., and Parker, Laurie L.
- Subjects
- *
CHRONIC myeloid leukemia , *PROTEIN-tyrosine kinase inhibitors , *BIOLOGICAL assay , *TARGETED drug delivery , *DRUG efficacy , *DIAGNOSIS - Abstract
Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
30. High Throughput Kinomic Profiling of Human Clear Cell Renal Cell Carcinoma Identifies Kinase Activity Dependent Molecular Subtypes.
- Author
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Anderson, Joshua C., Willey, Christopher D., Mehta, Amitkumar, Welaya, Karim, Chen, Dongquan, Duarte, Christine W., Ghatalia, Pooja, Arafat, Waleed, Madan, Ankit, Sudarshan, Sunil, Naik, Gurudatta, Grizzle, William E., Choueiri, Toni K., and Sonpavde, Guru
- Subjects
- *
RENAL cell carcinoma , *CANCER treatment , *PROTEIN kinases , *CANCER relapse , *MOLECULAR oncology , *HEALTH outcome assessment , *DIAGNOSIS - Abstract
Despite the widespread use of kinase-targeted agents in clear cell renal cell carcinoma (CC-RCC), comprehensive kinase activity evaluation (kinomic profiling) of these tumors is lacking. Thus, kinomic profiling of CC-RCC may assist in devising a classification system associated with clinical outcomes, and help identify potential therapeutic targets. Fresh frozen CC-RCC tumor lysates from 41 clinically annotated patients who had localized disease at diagnosis were kinomically profiled using the PamStation®12 high-content phospho-peptide substrate microarray system (PamGene International). Twelve of these patients also had matched normal kidneys available that were also profiled. Unsupervised hierarchical clustering and supervised comparisons based on tumor vs. normal kidney and clinical outcome (tumor recurrence) were performed and coupled with advanced network modeling and upstream kinase prediction methods. Unsupervised clustering analysis of localized CC-RCC tumors identified 3 major kinomic groups associated with inflammation (A), translation initiation (B), and immune response and cell adhesions (C) processes. Potential driver kinases implicated include PFTAIRE (PFTK1), PKG1, and SRC, which were identified in groups A, B, and C, respectively. Of the 9 patients who had tumor recurrence, only one was found in Group B. Supervised analysis showed decreased kinase activity of CDK1 and RSK1-4 substrates in those which progressed compared to others. Twelve tumors with matching normal renal tissue implicated increased PIM’s and MAPKAPK’s in tumors compared to adjacent normal renal tissue. As such, comprehensive kinase profiling of CC-RCC tumors could provide a functional classification strategy for patients with localized disease and identify potential therapeutic targets. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
31. Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src.
- Author
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Stateva, Silviya R., Salas, Valentina, Anguita, Estefanía, Benaim, Gustavo, and Villalobo, Antonio
- Subjects
- *
PROTEIN-tyrosine kinases , *CALMODULIN , *CALCIUM ions , *SRC gene , *CELL receptors , *CELL proliferation - Abstract
Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
32. The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals.
- Author
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Liu, Qian, Cho, Hoonsik, Yeo, Won-Sik, and Bae, Taeok
- Subjects
- *
STAPHYLOCOCCUS aureus , *MICROBIAL virulence , *HISTIDINE kinases , *PROTEIN kinases , *PATHOGENIC bacteria , *PEPTIDES - Abstract
Bacterial pathogens often employ two-component systems (TCSs), typically consisting of a sensor kinase and a response regulator, to control expression of a set of virulence genes in response to changing host environments. In Staphylococcus aureus, the SaeRS TCS is essential for in vivo survival of the bacterium. The intramembrane-sensing histidine kinase SaeS contains, along with a C-terminal kinase domain, a simple N-terminal domain composed of two transmembrane helices and a nine amino acid-long extracytoplasmic linker peptide. As a molecular switch, SaeS maintains low but significant basal kinase activity and increases its kinase activity in response to inducing signals such as human neutrophil peptide 1 (HNP1). Here we show that the linker peptide of SaeS controls SaeS’s basal kinase activity and that the amino acid sequence of the linker peptide is highly optimized for its function. Without the linker peptide, SaeS displays aberrantly elevated kinase activity even in the absence of the inducing signal, and does not respond to HNP1. Moreover, SaeS variants with alanine substitution of the linker peptide amino acids exhibit altered basal kinase activity and/or irresponsiveness to HNP1. Biochemical assays reveal that those SaeS variants have altered autokinase and phosphotransferase activities. Finally, animal experiments demonstrate that the linker peptide-mediated fine tuning of SaeS kinase activity is critical for survival of the pathogen. Our results indicate that the function of the linker peptide in SaeS is a highly evolved feature with very optimized amino acid sequences, and we propose that, in other SaeS-like intramembrane sensing histidine kinases, the extracytoplasmic linker peptides actively fine-control their kinases. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
33. SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products.
- Author
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Sowd, Gregory A., Mody, Dviti, Eggold, Joshua, Cortez, David, Friedman, Katherine L., and Fanning, Ellen
- Subjects
- *
KINASE genetics , *SIMIAN viruses , *DNA replication , *GENOMES , *CELL cycle - Abstract
Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
34. Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation.
- Author
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Vincent, Emma E., Elder, Douglas J. E., O′Flaherty, Linda, Pardo, Olivier E., Dzien, Piotr, Phillips, Lois, Morgan, Carys, Pawade, Joya, May, Margaret T., Sohail, Muhammad, Hetzel, Martin R., Seckl, Michael J., and Tavaré, Jeremy M.
- Subjects
- *
GLYCOGEN synthase kinase-3 , *PROTEIN kinases , *ENZYME activation , *NON-small-cell lung carcinoma , *CANCER cells , *TUMOR growth - Abstract
Background: Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC. Methods: The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture. Results: Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes. Conclusion: NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
35. Expression of Concern: High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity.
- Subjects
- *
MITOGEN-activated protein kinases , *CALCIUM , *OSCILLATIONS - Published
- 2023
- Full Text
- View/download PDF
36. Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation
- Author
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Emma E Vincent, Douglas J E Elder, Linda O'Flaherty, Olivier E Pardo, Piotr Dzien, Lois Phillips, Carys Morgan, Joya Pawade, Margaret T May, Muhammad Sohail, Martin R Hetzel, Michael J Seckl, and Jeremy M Tavaré
- Subjects
animal structures ,Lung Neoplasms ,Blotting, Western ,Cancer Treatment ,lcsh:Medicine ,Apoptosis ,macromolecular substances ,Biochemistry ,Lung and Intrathoracic Tumors ,Signaling Molecules ,Immunoenzyme Techniques ,Glycogen Synthase Kinase 3 ,Cell Signaling ,Carcinoma, Non-Small-Cell Lung ,Basic Cancer Research ,Medicine and Health Sciences ,Tumor Cells, Cultured ,Humans ,Post-Translational Modification ,Phosphorylation ,lcsh:Science ,Lung ,Chemotherapeutic Agents ,Cell Proliferation ,lcsh:R ,Biology and Life Sciences ,Proteins ,Cancers and Neoplasms ,Cell Biology ,respiratory tract diseases ,Non-Small Cell Lung Cancer ,Oncology ,Case-Control Studies ,lcsh:Q ,Oncology Agents ,Biomarkers ,Research Article ,Signal Transduction - Abstract
© 2014 Vincent et al.Background: Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC. Methods: The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture. Results: Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes. Conclusion: NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.
- Published
- 2014
37. High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity.
- Author
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Morita, Mitsuhiro, Nakane, Akira, Fujii, Yuki, Maekawa, Shohei, and Kudo, Yoshihisa
- Subjects
- *
CALCIUM , *MITOGEN-activated protein kinases , *HISTAMINE receptors , *HYDROLYSIS , *PHOSPHOLIPIDS - Abstract
Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 104/cm2 showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 104/cm2 largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
38. Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity.
- Author
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Worku N, Stich A, Daugschies A, Wenzel I, Kurz R, Thieme R, Kurz S, and Birkenmeier G
- Subjects
- Cell Proliferation drug effects, Cell Survival drug effects, Culture Media chemistry, Drug Delivery Systems methods, Drug Resistance, Enzyme Assays, Kinetics, Protozoan Proteins metabolism, Pyruvate Kinase metabolism, Trypanosoma brucei brucei enzymology, Trypanosoma brucei brucei growth & development, Protozoan Proteins antagonists & inhibitors, Pyruvate Kinase antagonists & inhibitors, Pyruvates pharmacology, Trypanocidal Agents pharmacology, Trypanosoma brucei brucei drug effects
- Abstract
Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties., Results: The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions., Conclusion: Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the blood-brain-barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemolymphatic as well as neurological stages of sleeping sickness.
- Published
- 2015
- Full Text
- View/download PDF
39. Regulation of Pancreatic β Cell Mass by Cross-Interaction between CCAAT Enhancer Binding Protein β Induced by Endoplasmic Reticulum Stress and AMP-Activated Protein Kinase Activity.
- Author
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Matsuda T, Takahashi H, Mieda Y, Shimizu S, Kawamoto T, Matsuura Y, Takai T, Suzuki E, Kanno A, Koyanagi-Kimura M, Asahara S, Bartolome A, Yokoi N, Inoue H, Ogawa W, Seino S, and Kido Y
- Subjects
- AMP-Activated Protein Kinases genetics, Adamantane analogs & derivatives, Adamantane pharmacology, Aminoimidazole Carboxamide analogs & derivatives, Aminoimidazole Carboxamide pharmacology, Animals, CCAAT-Enhancer-Binding Protein-beta genetics, Cell Line, Diabetes Mellitus, Type 2 metabolism, Diabetes Mellitus, Type 2 pathology, Gene Expression Regulation drug effects, Glucose Tolerance Test, Hypoglycemic Agents pharmacology, Insulin-Secreting Cells cytology, Insulin-Secreting Cells drug effects, Insulin-Secreting Cells metabolism, Islets of Langerhans metabolism, Islets of Langerhans pathology, Metformin pharmacology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Nitriles pharmacology, Phosphorylation drug effects, Pyrrolidines pharmacology, Ribonucleotides pharmacology, Vildagliptin, AMP-Activated Protein Kinases metabolism, CCAAT-Enhancer-Binding Protein-beta metabolism, Endoplasmic Reticulum Stress physiology
- Abstract
During the development of type 2 diabetes, endoplasmic reticulum (ER) stress leads to not only insulin resistance but also to pancreatic beta cell failure. Conversely, cell function under various stressed conditions can be restored by reducing ER stress by activating AMP-activated protein kinase (AMPK). However, the details of this mechanism are still obscure. Therefore, the current study aims to elucidate the role of AMPK activity during ER stress-associated pancreatic beta cell failure. MIN6 cells were loaded with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) and metformin to assess the relationship between AMPK activity and CCAAT enhancer binding protein β (C/EBPβ) expression levels. The effect of C/EBPβ phosphorylation on expression levels was also investigated. Vildagliptin and metformin were administered to pancreatic beta cell-specific C/EBPβ transgenic mice to investigate the relationship between C/EBPβ expression levels and AMPK activity in the pancreatic islets. When pancreatic beta cells are exposed to ER stress, the accumulation of the transcription factor C/EBPβ lowers the AMP/ATP ratio, thereby decreasing AMPK activity. In an opposite manner, incubation of MIN6 cells with AICAR or metformin activated AMPK, which suppressed C/EBPβ expression. In addition, administration of the dipeptidyl peptidase-4 inhibitor vildagliptin and metformin to pancreatic beta cell-specific C/EBPβ transgenic mice decreased C/EBPβ expression levels and enhanced pancreatic beta cell mass in proportion to the recovery of AMPK activity. Enhanced C/EBPβ expression and decreased AMPK activity act synergistically to induce ER stress-associated pancreatic beta cell failure.
- Published
- 2015
- Full Text
- View/download PDF
40. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.
- Author
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Lageix S, Zhang J, Rothenburg S, and Hinnebusch AG
- Subjects
- Amino Acid Substitution genetics, Crystallography, X-Ray, Eukaryotic Initiation Factor-2 genetics, Histidine-tRNA Ligase chemistry, Mutation, Phosphorylation, Protein Conformation, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases metabolism, Protein Structure, Tertiary, RNA, Transfer metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Trypanosoma cruzi, Histidine-tRNA Ligase genetics, Protein Serine-Threonine Kinases genetics, RNA, Transfer genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.
- Published
- 2015
- Full Text
- View/download PDF
41. Ethyl pyruvate emerges as a safe and fast acting agent against Trypanosoma brucei by targeting pyruvate kinase activity
- Author
-
Netsanet, Worku, August, Stich, Arwid, Daugschies, Iris, Wenzel, Randy, Kurz, Rene, Thieme, Susanne, Kurz, Gerd, Birkenmeier, Universität Leipzig, Missionsärztliches Institut Würzburg, and Universitätsklinikum Leipzig
- Subjects
ddc:616 ,Cell Survival ,Pyruvate Kinase ,Trypanosoma brucei brucei ,lcsh:R ,Drug Resistance ,Protozoan Proteins ,lcsh:Medicine ,Afrikanische Trypanosomiasis, Tropenerkrankheit, Trypanosoma brucei ,Trypanocidal Agents ,Culture Media ,Human African Trypanosomiasis (HAT), tropical disease, Trypanosoma brucei ,Kinetics ,Drug Delivery Systems ,lcsh:Q ,ddc:610 ,Pyruvates ,lcsh:Science ,Cell Proliferation ,Enzyme Assays ,Research Article - Abstract
Background Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. Results The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0\(\pm\)0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. Conclusion Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the blood-brain-barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemo-lymphatic as well as neurological stages of sleeping sickness.
- Published
- 2015
42. Pharmacologic inhibition of MLK3 kinase activity blocks the in vitro migratory capacity of breast cancer cells but has no effect on breast cancer brain metastasis in a mouse xenograft model.
- Author
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Rhoo KH, Granger M, Sur J, Feng C, Gelbard HA, Dewhurst S, and Polesskaya O
- Subjects
- Animals, Cell Line, Tumor, Disease Models, Animal, Female, Mice, Mice, Nude, Neoplasm Transplantation, Protein Kinase Inhibitors pharmacology, Pyridines pharmacology, Pyrroles pharmacology, Transplantation, Heterologous, Mitogen-Activated Protein Kinase Kinase Kinase 11, Brain Neoplasms secondary, Breast Neoplasms pathology, Cell Movement drug effects, MAP Kinase Kinase Kinases antagonists & inhibitors
- Abstract
Brain metastasis of breast cancer is an important clinical problem, with few therapeutic options and a poor prognosis. Recent data have implicated mixed lineage kinase 3 (MLK3) in controlling the in vitro migratory capacity of breast cancer cells, as well as the metastasis of MDA-MB-231 breast cancer cells from the mammary fat pad to distant lymph nodes in a mouse xenograft model. We therefore set out to test whether MLK3 plays a role in brain metastasis of breast cancer cells. To address this question, we used a novel, brain penetrant, MLK3 inhibitor, URMC099. URMC099 efficiently inhibited the migration of breast cancer cells in an in vitro cell monolayer wounding assay, and an in vitro transwell migration assay, but had no effect on in vitro cell growth. We also tested the effect of URMC099 on tumor formation in a mouse xenograft model of breast cancer brain metastasis. This analysis showed that URMC099 had no effect on the either the frequency or size of breast cancer brain metastases. We conclude that pharmacologic inhibition of MLK3 by URMC099 can reduce the in vitro migratory capacity of breast cancer cells, but that it has no effect on either the frequency or size of breast cancer brain metastases, in a mouse xenograft model.
- Published
- 2014
- Full Text
- View/download PDF
43. Cardiac-specific inhibition of kinase activity in calcium/calmodulin-dependent protein kinase kinase-β leads to accelerated left ventricular remodeling and heart failure after transverse aortic constriction in mice.
- Author
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Watanabe S, Horie T, Nagao K, Kuwabara Y, Baba O, Nishi H, Sowa N, Narazaki M, Matsuda T, Takemura G, Wada H, Hasegawa K, Kimura T, and Ono K
- Subjects
- Adenosine Triphosphate, Animals, Calcium-Calmodulin-Dependent Protein Kinase Kinase metabolism, Disease Models, Animal, Gene Expression Regulation, Heart Failure metabolism, Heart Failure mortality, Heart Failure physiopathology, Heart Ventricles metabolism, Heart Ventricles pathology, Magnetic Resonance Spectroscopy, Male, Mice, Mice, Transgenic, Mitochondria, Heart genetics, Mitochondria, Heart metabolism, Myosin Heavy Chains genetics, Phosphorylation, Promoter Regions, Genetic, Signal Transduction, Up-Regulation, Ventricular Dysfunction, Left genetics, Ventricular Dysfunction, Left physiopathology, Calcium-Calmodulin-Dependent Protein Kinase Kinase genetics, Heart Failure etiology, Ventricular Remodeling genetics
- Abstract
Background: The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated., Methods and Results: We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) transgenic (α-MHC CaMKKβkd TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKβ activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKβkd TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKβ downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKβkd TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKβ and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKβkd TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/β-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKβkd TG mice compared with WT mice after TAC., Conclusions: These data indicate that CaMKKβ exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKβ may be a therapeutic target for the treatment of heart failure.
- Published
- 2014
- Full Text
- View/download PDF
44. Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.
- Author
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Koussis, Konstantinos, Haase, Silvia, Withers-Martinez, Chrislaine, Flynn, Helen R., Kunzelmann, Simone, Christodoulou, Evangelos, Ibrahim, Fairouz, Skehel, Mark, Baker, David A., and Blackman, Michael J.
- Subjects
PLASMODIUM ,CGMP-dependent protein kinase ,PHOSPHORYLATION ,PARASITE life cycles ,ERYTHROCYTES ,SERINE/THREONINE kinases ,MALARIA prevention - Abstract
The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development. Author summary: Despite all efforts to control and eradicate malaria, the disease still poses a huge burden on human health. Almost half of the world's population lives in high malaria transmission areas with over half a million deaths occurring annually due to the disease, which is caused by a single-celled parasite called Plasmodium. Replication of the parasite inside red blood cells is responsible for all the clinical manifestations of the disease. At the end of each replicative cycle, parasites rupture the red blood cell in a process known as egress in order to invade new red blood cells. Previous studies have shown that an essential kinase termed PKG is a master regulator of egress. However, many aspects of PKG regulation are still unknown. In this work we examined the importance of phosphorylation on PKG function by replacing wild type PKG with mutant forms refractory to phosphorylation. We found that phosphorylation in a specific region of the protein is essential for parasite survival. Excitingly though, phosphorylation is not essential for kinase activity, as has been shown for mammalian PKG proteins but regulates its substrate specificity. Our results suggest that Plasmodium PKG is uniquely regulated compared to its mammalian counterparts, to facilitate parasite proliferation. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
45. Novel hematopoietic progenitor kinase 1 inhibitor KHK-6 enhances T-cell activation.
- Author
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Ahn, Min Jeong, Kim, Eun Hye, Choi, Yunha, Chae, Chong Hak, Kim, Pilho, and Kim, Seong Hwan
- Subjects
T cells ,KINASE inhibitors ,DRUG discovery ,CANCER cells ,IMMUNE response ,HLA-DR antigens - Abstract
Inhibiting the functional role of negative regulators in immune cells is an effective approach for developing immunotherapies. The serine/threonine kinase hematopoietic progenitor kinase 1 (HPK1) involved in the T-cell receptor signaling pathway attenuates T-cell activation by inducing the degradation of SLP-76 through its phosphorylation at Ser-376, reducing the immune response. Interestingly, several studies have shown that the genetic ablation or pharmacological inhibition of HPK1 kinase activity improves the immune response to cancers by enhancing T-cell activation and cytokine production; therefore, HPK1 could be a promising druggable target for T-cell-based cancer immunotherapy. To increase the immune response against cancer cells, we designed and synthesized KHK-6 and evaluated its cellular activity to inhibit HPK1 and enhance T-cell activation. KHK-6 inhibited HPK1 kinase activity with an IC
50 value of 20 nM and CD3/CD28-induced phosphorylation of SLP-76 at Ser-376 Moreover, KHK-6 significantly enhanced CD3/CD28-induced production of cytokines; proportion of CD4+ and CD8+ T cells that expressed CD69, CD25, and HLA-DR markers; and T-cell-mediated killing activity of SKOV3 and A549 cells. In conclusion, KHK-6 is a novel ATP-competitive HPK1 inhibitor that blocks the phosphorylation of HPK1 downstream of SLP-76, enhancing the functional activation of T cells. In summary, our study showed the usefulness of KHK-6 in the drug discovery for the HPK1-inhibiting immunotherapy. [ABSTRACT FROM AUTHOR]- Published
- 2024
- Full Text
- View/download PDF
46. The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.
- Author
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Czajkowski, Emily R., Zou, Yuntong, Divekar, Nikita S., and Wignall, Sarah M.
- Subjects
GERM cells ,PARTITION functions ,CAENORHABDITIS elegans ,MICROTUBULE-associated proteins ,CELL division ,CHROMOSOME segregation ,MICROTUBULES - Abstract
Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis. Author summary: When a cell divides, a football-shaped spindle made up of dynamic microtubule polymers functions to partition chromosomes. Usually, structures called centrosomes organize microtubules at the two ends of the spindle. However, in female reproductive cells ("oocytes") of many organisms, the spindle is formed without the help of centrosomes. In this study, we used the model organism C. elegans to understand how this type of "acentrosomal" spindle forms and maintains its shape. Specifically, we investigated a protein called ZYG-8 and we found that when this protein was removed from oocytes, the spindles became overelongated and their typical football shape was disrupted. Follow-up studies revealed that ZYG-8 plays two important functions that promote acentrosomal spindle assembly and stability in oocytes: 1) ZYG-8 helps control the dynamic properties of the microtubule polymers so that they can be properly arranged into the spindle structure and 2) ZYG-8 tunes forces within the spindle, to help the structure maintain it shape. Taken together, our findings have increased our understanding of how spindles form in the absence of centrosomes, shedding light on how female reproductive cells divide. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
47. Blockade of IKK signaling induces RIPK1-independent apoptosis in human macrophages.
- Author
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Nataraj, Neha M., Sillas, Reyna Garcia, Herrmann, Beatrice I., Shin, Sunny, and Brodsky, Igor E.
- Subjects
APOPTOSIS ,YERSINIA pseudotuberculosis ,CELL death ,GRAM-negative bacteria ,SECRETION ,YERSINIA - Abstract
Regulated cell death in response to microbial infection plays an important role in immune defense and is triggered by pathogen disruption of essential cellular pathways. Gram-negative bacterial pathogens in the Yersinia genus disrupt NF-κB signaling via translocated effectors injected by a type III secretion system, thereby preventing induction of cytokine production and antimicrobial defense. In murine models of infection, Yersinia blockade of NF-κB signaling triggers cell-extrinsic apoptosis through Receptor Interacting Serine-Threonine Protein Kinase 1 (RIPK1) and caspase-8, which is required for bacterial clearance and host survival. Unexpectedly, we find that human macrophages undergo apoptosis independently of RIPK1 in response to Yersinia or chemical blockade of IKKβ. Instead, IKK blockade led to decreased cFLIP expression, and overexpression of cFLIP contributed to protection from IKK blockade-induced apoptosis in human macrophages. We found that IKK blockade also induces RIPK1 kinase activity-independent apoptosis in human T cells and human pancreatic cells. Altogether, our data indicate that, in contrast to murine cells, blockade of IKK activity in human cells triggers a distinct apoptosis pathway that is independent of RIPK1 kinase activity. These findings have implications for the contribution of RIPK1 to cell death in human cells and the efficacy of RIPK1 inhibition in human diseases. Author summary: Programmed cell death is critical for organismal homeostasis and for host defense against microbial infection. Gram-negative bacteria of the genus Yersinia cause diseases ranging from plague (Y. pestis) to severe gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica). Studies in murine models have demonstrated that all pathogenic Yersinia disrupt pro-inflammatory cell signaling, which triggers cell death in murine macrophages. This cell death is mediated by the kinase RIPK1, and RIPK1-mediated cell death is essential for host defense and survival. Although murine studies have provided insight into mechanisms that regulate host defense during Yersinia infection, there are important differences between human and murine immune systems regarding expression and presence of key proteins. Thus, how human cells respond to Yersinia infection remains poorly understood. Here, we report that, in contrast to murine systems, Yersinia infection or chemical blockade of immune signaling in human cells induces a distinct apoptotic cell death that is entirely independent of RIPK1 kinase activity. RIPK1 is implicated in a wide range of systemic disorders and is currently being targeted in clinical trials. Our study provides insight into human-specific cell death signaling and host defense mechanisms, which has implications for understanding human immune responses to infection and therapeutic approaches for treating human diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
48. Genome-wide CRISPR/Cas9 screen identifies SLC39A9 and PIK3C3 as crucial entry factors for Ebola virus infection.
- Author
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Gong, Mingli, Peng, Cheng, Yang, Chen, Wang, Zhenhua, Qian, Hongwu, Hu, Xue, Zhou, Peng, Shan, Chao, and Ding, Qiang
- Subjects
EBOLA virus disease ,PHOSPHATIDYLINOSITOL 3-kinases ,VIRAL proteins ,RECOMBINANT proteins ,EBOLA virus - Abstract
The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013–2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection. Author summary: Ebola virus (EBOV) remains a significant global health threat. The limited availability of high-containment (BSL-4) facilities hinders our understanding of the interaction of virus with host cells and the development of effective treatments. To address this, we utilized a biologically contained EBOVΔVP30-EGFP model to identify critical host factors necessary for EBOV infection through a genome-wide CRISPR/Cas9 screen conducted at BSL-2 lab. Our study revealed that two proteins, PIK3C3 and SLC39A9, are essential for the virus to enter host cells. We found that PIK3C3 is required for EBOV endosomal trafficking, and inhibiting its kinase activity can significantly reduce infection, making it a promising target for antiviral therapy. Additionally, we discovered that SLC39A9 facilitates viral entry by interacting with the viral glycoprotein (GP) and enhancing its binding to the cell's NPC1 receptor, rather than through its usual role in zinc transport. These findings offer new insights into how EBOV enters cells and highlight potential targets for developing therapies to combat Ebola virus infection. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
49. A degron-based strategy reveals new insights into Aurora B function in C. elegans.
- Author
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Divekar, Nikita S., Davis-Roca, Amanda C., Zhang, Liangyu, Dernburg, Abby F., and Wignall, Sarah M.
- Subjects
CELL division ,CAENORHABDITIS elegans ,AURORA kinases ,CHROMOSOME segregation ,CELL cycle ,B cells - Abstract
The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2's major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators. Author summary: During cell division, chromosomes must be accurately partitioned to ensure the proper distribution of genetic material. In mitosis, chromosomes are duplicated once and then divided once, generating daughter cells with the same amount of genetic material as the original cell. Conversely, during meiosis chromosomes are duplicated once and divided twice, to cut the chromosome number in half to generate eggs and sperm. One important protein that is required for both mitotic and meiotic chromosome segregation is the kinase Aurora B, which phosphorylates a variety of other cell division proteins. However, previous research has shown that some kinases have functions that are independent of their ability to phosphorylate other proteins. Thus, fully understanding how Aurora B regulates cell division requires methods to test whether its various functions require kinase activity. We designed and implemented such a strategy in the model organism C. elegans, by depleting Aurora B from meiotically and mitotically-dividing cells, leaving in place a kinase-inactive version. This work has lent insight into how Aurora B regulates cell division in C. elegans, and also serves as a proof of principle for our approach, which can now be applied to study other essential cell division kinases. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
50. Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system.
- Author
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Yamamoto, Naoki, Kikuchi, Jiro, Furukawa, Yusuke, and Shibayama, Naoya
- Subjects
PROTEIN expression ,PROTEIN-tyrosine kinase inhibitors ,SILKWORMS ,PROTEIN-tyrosine kinases ,PUPAE ,KINASE inhibitors - Abstract
We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 kinase inhibitors, and succeeded in screening two inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
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