8,434 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
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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. Oncogenic mutations of p110α isoform of PI 3-kinase upregulate its protein kinase activity
- Author
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Buchanan, Christina M., Dickson, James M. J., Lee, Woo-Jeong, Guthridge, Mark A., Kendall, Jackie D., Shepherd, Peter R., Buchanan, Christina M., Dickson, James M. J., Lee, Woo-Jeong, Guthridge, Mark A., Kendall, Jackie D., and Shepherd, Peter R.
- Abstract
In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 βc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 βc receptor (βic). We find p110α, p110β and p110γ all phosphorylate βic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards βic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn2+ dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110β and p110γ all display significant activity in the presence of Mg2+. Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.
- Published
- 2013
10. Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival
- Author
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Thomas, Daniel, Powell, Jason A., Green, Benjamin D., Barry, Emma F., Ma, Yuefang, Woodcock, Joanna, Fitter, Stephen, Zannettino, Andrew C. W., Pitson, Stuart M., Hughes, Timothy P., Lopez, Angel F., Shepherd, Peter R., Wei, Andrew H., Ekert, Paul G., Guthridge, Mark A., Thomas, Daniel, Powell, Jason A., Green, Benjamin D., Barry, Emma F., Ma, Yuefang, Woodcock, Joanna, Fitter, Stephen, Zannettino, Andrew C. W., Pitson, Stuart M., Hughes, Timothy P., Lopez, Angel F., Shepherd, Peter R., Wei, Andrew H., Ekert, Paul G., and Guthridge, Mark A.
- Abstract
The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathway
- Published
- 2013
11. 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
- Full Text
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12. 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
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13. 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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14. 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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15. 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
- Full Text
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16. RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development
- Author
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Magariños, Marta, Rodríguez Aburto, María, Sánchez-Calderón, Hortensia, Muñoz Agudo, Carmen, Rapp, Ulf R., Varela-Nieto, Isabel, Magariños, Marta, Rodríguez Aburto, María, Sánchez-Calderón, Hortensia, Muñoz Agudo, Carmen, Rapp, Ulf R., and Varela-Nieto, Isabel
- Abstract
[Background]: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. [Methodology/Principal Findings]: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. [Conclusions/Significance]: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.
- Published
- 2010
17. Ethyl pyruvate emerges as a safe and fast acting agent against Trypanosoma brucei by targeting pyruvate kinase activity
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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
18. 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
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- *
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]
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- 2019
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19. Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity
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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.
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- 2016
20. MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK
- Author
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Zhang, Gen, He, Li-Sheng, Wong, Yue Him, Qian, Pei-Yuan, Zhang, Gen, He, Li-Sheng, Wong, Yue Him, and Qian, Pei-Yuan
- Abstract
The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway. © 2013 Zhang et al.
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- 2013
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
- Author
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尾野, 亘, 20823082, 20565577, 60632099, 00209561, 80359786, Watanabe, Shin, Horie, Takahiro, Nagao, Kazuya, Kuwabara, Yasuhide, Baba, Osamu, Nishi, Hitoo, Sowa, Naoya, Narazaki, Michiko, Matsuda, Tetsuya, Takemura, Genzou, Wada, Hiromichi, Hasegawa, Koji, Kimura, Takeshi, Ono, Koh, 尾野, 亘, 20823082, 20565577, 60632099, 00209561, 80359786, Watanabe, Shin, Horie, Takahiro, Nagao, Kazuya, Kuwabara, Yasuhide, Baba, Osamu, Nishi, Hitoo, Sowa, Naoya, Narazaki, Michiko, Matsuda, Tetsuya, Takemura, Genzou, Wada, Hiromichi, Hasegawa, Koji, Kimura, Takeshi, and Ono, Koh
- 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. Ca2+/calmodulin and apo-calmodulin both bind to and enhance the tyrosine kinase activity of c-Src
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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
23. 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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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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24. SpdC, a novel virulence factor, controls histidine kinase activity in Staphylococcus aureus.
- Author
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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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25. Protein-tyrosine kinase activity profiling in knock down zebrafish embryos
- Author
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Biomoleculaire Massaspectrometrie, Massaspectrometrie, SYNTHESE, Dep Farmaceutische wetenschappen, Dep Scheikunde, Lemeer, S.M., Jopling, J.C., Ruijtenbeek, R., Slijper, M., Heck, A.J.R., den Hertog, J., Biomoleculaire Massaspectrometrie, Massaspectrometrie, SYNTHESE, Dep Farmaceutische wetenschappen, Dep Scheikunde, Lemeer, S.M., Jopling, J.C., Ruijtenbeek, R., Slijper, M., Heck, A.J.R., and den Hertog, J.
- Published
- 2007
26. 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
- Subjects
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
27. 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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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
28. 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]
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- 2017
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29. 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.
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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
30. IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation
- Author
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Annalisa Astolfi, Mary B. Goldring, Stefania Pagani, Rosa Maria Borzì, Andrea Facchini, Eleonora Olivotto, Flavio Flamigni, Annalisa Facchini, Kenneth B. Marcu, Daniela Platano, Miguel Otero, Eleonora Olivotto, Miguel Otero, Annalisa Astolfi, Daniela Platano, Annalisa Facchini, Stefania Pagani, Flavio Flamigni, Andrea Facchini, Mary B. Goldring, Rosa Maria Borzì, and Kenneth B. Marcu
- Subjects
Cellular differentiation ,Immunoblotting ,lcsh:Medicine ,Biology ,Chondrocyte ,Extracellular matrix ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Chondrocytes ,Matrix Metalloproteinase 10 ,Matrix Metalloproteinase 13 ,medicine ,Animals ,Humans ,Kinase activity ,lcsh:Science ,CHUK ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Kinase ,Effector ,Reverse Transcriptase Polymerase Chain Reaction ,lcsh:R ,I-Kappa-B Kinase ,Cell Differentiation ,Molecular biology ,Immunohistochemistry ,Cell biology ,Extracellular Matrix ,I-kappa B Kinase ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,lcsh:Q ,NF-KAPPA-B, METALLOPROTEINASE 13-DEFICIENT MICE, HUMAN OSTEOARTHRITIC CHONDROCYTES, HELIX-LOOP-HELIX, GENE-EXPRESSION, GROWTH-PLATE, TRANSCRIPTIONAL CONTROL, II COLLAGEN, CARTILAGE DEGENERATION, CELL-DIFFERENTIATION ,Research Article - Abstract
Background: The non-canonical NF-kappa B activating kinase IKK alpha, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti-inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKK alpha as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy. Methodology/Principal Findings: IKK alpha expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKK alpha(f/f):CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKK alpha in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKK alpha ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKK alpha-deficient chondrocytes. IKK alpha deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of. Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKK alpha-deficient chondrocytes was rescued by a kinase-dead IKK alpha protein mutant. Conclusions/Significance: IKK alpha acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKK alpha positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. T eta nu sigma, our findings are also of potential relevance for defining the onset and/or progression of OA disease.
- Published
- 2013
31. A Series of Beta-Carboline Derivatives Inhibit the Kinase Activity of PLKs
- Author
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Ni Li, Jing Zhang, Shuyi Si, Xiao-Min Han, Jialin Wu, Yongzhen Li, Yanchang Wang, Liang Guo, Qin Ma, and Rihui Cao
- Subjects
Cancer Treatment ,lcsh:Medicine ,Apoptosis ,Cell Cycle Proteins ,Biochemistry ,HeLa ,0302 clinical medicine ,Drug Discovery ,Molecular Cell Biology ,Basic Cancer Research ,Signaling in Cellular Processes ,lcsh:Science ,Apoptotic Signaling Cascade ,Apoptotic Signaling ,0303 health sciences ,Multidisciplinary ,biology ,Kinase ,Cell Cycle ,Cell cycle ,Signaling Cascades ,3. Good health ,Cell biology ,Wee1 ,Oncology ,030220 oncology & carcinogenesis ,Medicine ,Research Article ,Biotechnology ,Signal Transduction ,Drugs and Devices ,Drug Research and Development ,Protein Serine-Threonine Kinases ,PLK1 ,03 medical and health sciences ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Humans ,Kinase activity ,Mitosis ,Biology ,Protein Kinase Inhibitors ,030304 developmental biology ,lcsh:R ,Chemotherapy and Drug Treatment ,biology.organism_classification ,Cancer cell ,biology.protein ,lcsh:Q ,Carbolines - Abstract
Polo-like kinases play an essential role in the ordered execution of mitotic events and 4 mammalian PLK family members have been identified. Accumulating evidence indicates that PLK1 is an attractive target for anticancer drugs. In this paper, a series of beta-carboline derivatives were synthesized and three compounds, DH281, DH285 and DH287, were identified as potent new PLK inhibitors. We employed various biochemical and cellular approaches to determine the effects of these compounds on the activity of PLK1 and other mitotic kinases and on cell cycle progression. We found that these three compounds could selectively inhibit the kinase activity of purified PLK1, PLK2 and PLK3 in vitro. They show strong antitumor activity against a number of cancer cell lines with relatively low micromolar IC(50)s, but are relatively less toxic to non-cancer cells (MRC5). Moreover, these compounds could induce obvious accumulation of HeLa cells in G(2)/M and S phases and trigger apoptosis. Although MRC5 cells show clear S-phase arrest after treatment with these compounds, the G2/M arrest and apoptosis are less insignificant, indicating the distinct sensitivity between normal and cancer cells. We also found that HeLa cells treated with these drugs exhibit monopolar spindles and increased Wee1 protein levels, the characteristics of cells treated with PLK1 inhibitors. Together, these results demonstrate that DH281, DH285 and DH287 beta-carboline compounds are new PLK inhibitors with potential for cancer treatment.
- Published
- 2012
32. 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
- Subjects
- 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.
- Published
- 2017
- Full Text
- View/download PDF
33. 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
34. Increased nucleoside diphosphate kinase activity induces white spot syndrome virus infection in Litopenaeus vannamei.
- Author
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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.
- Published
- 2017
- Full Text
- View/download PDF
35. 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]
- Published
- 2016
- Full Text
- View/download PDF
36. 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]
- Published
- 2016
- Full Text
- View/download PDF
37. A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.
- Author
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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
38. 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
39. 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
40. 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
41. 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
42. Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation.
- Author
-
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
43. 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
44. Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation
- Author
-
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
45. High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity.
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Morita, Mitsuhiro, Nakane, Akira, Fujii, Yuki, Maekawa, Shohei, and Kudo, Yoshihisa
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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]
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- 2015
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46. Inhibition of c-Abl Kinase Activity Renders Cancer Cells Highly Sensitive to Mitoxantrone.
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Alpay, Kemal, Farshchian, Mehdi, Tuomela, Johanna, Sandholm, Jouko, Aittokallio, Kaappo, Siljamäki, Elina, Kallio, Marko, Kähäri, Veli-Matti, and Hietanen, Sakari
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ABL1 gene , *MITOXANTRONE , *CANCER cells , *DNA damage , *CANCER chemotherapy , *PAPILLOMAVIRUSES - Abstract
Although c-Abl has increasingly emerged as a key player in the DNA damage response, its role in this context is far from clear. We studied the effect of inhibition of c-Abl kinase activity by imatinib with chemotherapy drugs and found a striking difference in cell survival after combined mitoxantrone (MX) and imatinib treatment compared to a panel of other chemotherapy drugs. The combinatory treatment induced apoptosis in HeLa cells and other cancer cell lines but not in primary fibroblasts. The difference in MX and doxorubicin was related to significant augmentation of DNA damage. Transcriptionally active p53 accumulated in cells in which human papillomavirus E6 normally degrades p53. The combination treatment resulted in caspase activation and apoptosis, but this effect did not depend on either p53 or p73 activity. Despite increased p53 activity, the cells arrested in G2 phase became defective in this checkpoint, allowing cell cycle progression. The effect after MX treatment depended partially on c-Abl: Short interfering RNA knockdown of c-Abl rendered HeLa cells less sensitive to MX. The effect of imatinib was decreased by c-Abl siRNA suggesting a role for catalytically inactive c-Abl in the death cascade. These findings indicate that MX has a unique cytotoxic effect when the kinase activity of c-Abl is inhibited. The treatment results in increased DNA damage and c-Abl–dependent apoptosis, which may offer new possibilities for potentiation of cancer chemotherapy. [ABSTRACT FROM AUTHOR]
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- 2014
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47. Lack of Correlation between the Kinase Activity of LRRK2 Harboring Kinase-Modifying Mutations and Its Phosphorylation at Ser910, 935, and Ser955.
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Ito, Genta, Fujimoto, Tetta, Kamikawaji, Shogo, Kuwahara, Tomoki, and Iwatsubo, Takeshi
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DARDARIN , *GENETIC mutation , *PHOSPHORYLATION , *GENETIC regulation , *ENZYME kinetics , *CELLULAR signal transduction , *NEURODEGENERATION - Abstract
Leucine-rich repeat kinase 2 (LRRK2) is extensively phosphorylated in cells within a region amino-terminal to the leucine-rich repeat domain. Since phosphorylation in this region of LRRK2, including Ser910, Ser935, Ser955, and Ser973, is significantly downregulated upon treatment with inhibitors of LRRK2, it has been hypothesized that signaling pathways downstream of the kinase activity of LRRK2 are involved in regulating the phosphorylation of LRRK2, although the precise mechanism has remained unknown. Here we examined the effects of LRRK2 inhibitors on the phosphorylation state at Ser910, Ser935, and Ser955 in a series of kinase-inactive mutants of LRRK2. We found that the responses of LRRK2 to the inhibitors varied among mutants, in a manner not consistent with the above-mentioned hypothesis. Notably, one of the kinase-inactive mutants, T2035A LRRK2, underwent phosphorylation, as well as the inhibitor-induced dephosphorylation, at Ser910, Ser935, and Ser955, to a similar extent to those observed with wild-type LRRK2. These results suggest that the kinase activity of LRRK2 is not involved in the common mechanism of inhibitor-induced dephosphorylation of LRRK2. [ABSTRACT FROM AUTHOR]
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- 2014
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48. Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions.
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Bøgebo, Rikke, Horn, Heiko, Olsen, Jesper V., Gammeltoft, Steen, Jensen, Lars J., Hansen, Jakob L., and Christensen, Gitte L.
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ANGIOTENSIN receptors , *PHOSPHOPROTEINS , *CELLULAR signal transduction , *BIOCHEMISTRY , *PROTEOMICS , *COMPUTATIONAL biology - Abstract
Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets. [ABSTRACT FROM AUTHOR]
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- 2014
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49. Microtubules Accelerate the Kinase Activity of Aurora-B by a Reduction in Dimensionality.
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Noujaim, Michael, Bechstedt, Susanne, Wieczorek, Michal, and Brouhard, Gary J.
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Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a ‘‘reduction in dimensionality.’’ We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates. [ABSTRACT FROM AUTHOR]
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- 2014
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50. Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone.
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Sharma, Poonam, Stagge, Stefan, Bekker, Martijn, Bettenbrock, Katja, and Hellingwerf, Klaas J.
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KINASES , *UBIQUINONES , *VITAMIN K2 , *GENETIC regulation , *GENE expression in bacteria , *BIOAVAILABILITY , *CYSTEINE , *ESCHERICHIA coli - Abstract
Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
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