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Mutations in signal transduction pathways lead to various diseases including cancers. MEK1 kinase, encoded by the human MAP2K1 gene, is one of the central components of the MAPK pathway and more than a hundred somatic mutations in the MAP2K1 gene were identified in various tumors. Germline mutations deregulating MEK1 also lead to congenital abnormalities, such as the cardiofaciocutaneous syndrome and arteriovenous malformation. Evaluating variants associated with a disease is a challenge, and computational genomic approaches aid in this process. Establishing evolutionary history of a gene improves computational prediction of disease-causing mutations; however, the evolutionary history of MEK1 is not well understood. Here, by revealing a precise evolutionary history of MEK1, we construct a well-defined dataset of MEK1 metazoan orthologs, which provides sufficient depth to distinguish between conserved and variable amino acid positions. We matched known and predicted disease-causing and benign mutations to evolutionary changes observed in corresponding amino acid positions and found that all known and many suspected disease-causing mutations are evolutionarily intolerable. We selected several variants that cannot be unambiguously assessed by automated prediction tools but that are confidently identified as "damaging" by our approach, for experimental validation in Drosophila. In all cases, evolutionary intolerant variants caused increased mortality and severe defects in fruit fly embryos confirming their damaging nature. We anticipate that our analysis will serve as a blueprint to help evaluate known and novel missense variants in MEK1 and that our approach will contribute to improving automated tools for disease-associated variant interpretation. [ABSTRACT FROM AUTHOR]

Phytophthora root and stem rot is a yield-limiting soybean disease caused by the soil-borne oomycete Phytophthora sojae. Although multiple quantitative disease resistance loci (QDRL) have been identified, most explain <10% of the phenotypic variation (PV). The major QDRL explaining up to 45% of the PV were previously identified on chromosome 18 and represent a valuable source of resistance for soybean breeding programs. Resistance alleles from plant introductions 427105B and 427106 significantly increase yield in disease-prone fields and result in no significant yield difference in fields with less to no disease pressure. In this study, high-resolution mapping reduced the QDRL interval to 3.1 cm, and RNA-seq analysis of near-isogenic lines (NILs) varying at QDRL-18 pinpointed a single gene of interest which was downregulated in inoculated NILs carrying the resistant allele compared to inoculated NILs with the susceptible allele. This gene of interest putatively encodes a serine–threonine kinase (STK) related to the AtCR4 family and may be acting as a susceptibility factor, based on the specific increase of jasmonic acid concentration in inoculated NILs. This work facilitates further functional analyses and marker-assisted breeding efforts by prioritizing candidate genes and narrowing the targeted region for introgression. [ABSTRACT FROM AUTHOR]

Phytophthora root and stem rot is a yield-limiting soybean disease caused by the soil-borne oomycete Phytophthora sojae. Although multiple quantitative disease resistance loci (QDRL) have been identified, most explain

Constitutive activation of FGFR1, as a result of diverse chromosome translocations, is the hallmark of stem cell leukemia/lymphoma syndrome. The BCR‐FGFR1 variant is unique in that the BCR component contributes a serine–threonine kinase (STK) to the N‐terminal end of the chimeric FGFR1 kinase. We have deleted the STK domain and mutated the critical Y177 residue and demonstrate that the transforming activity of these mutated genes is reduced compared to the BCR‐FGFR1 parental kinase. In addition, we demonstrate that deletion of the FGFR1 tyrosine kinase domain abrogates transforming ability, which is not compensated for by BCR STK activity. Unbiased screening for proteins that are inactivated as a result of loss of the BCR STK identified activated S6 kinase and SHP2 kinase. Genetic and pharmacological inhibition of SHP2 function in SCLL cells expressing BCR‐FGFR1 in vitro leads to reduced viability and increased apoptosis. In vivo treatment of SCLL in mice with SHP099 leads to suppression of leukemogenesis, supporting an important role for SHP2 in FGFR1‐driven leukemogenesis. In combination with the BGJ398 FGFR1 inhibitor, cell viability in vitro is further suppressed and acts synergistically with SHP099 in vivo suggesting a potential combined targeted therapy option in this subtype of SCLL disease. What's new? Constitutive activation of FGFR1 tyrosine kinase, as a result of diverse chromosome translocations, is the hallmark of stem cell leukemia/lymphoma syndrome. The relative contribution of the fusion partner genes remains unclear, however. Here, the authors show that the BCR gene serine–threonine kinase can modify disease course in vivo, through activation of SHP2 and its downstream targets. Pharmacologically targeting both SHP2 and FGFR1 displays synergistic efficacy in treating BCR‐FGFR1 disease subtype. The study indicates the potential role of fusion partner genes in modifying disease phenotype and rationalizes the need for precision therapeutics that target activation of both FGFR1 and fusion partners. [ABSTRACT FROM AUTHOR]

The pyrin inflammasome has evolved as an innate immune sensor to detect bacterial toxin-induced Rho guanosine triphosphatase (Rho GTPase)-inactivation, a process that is similar to the “guard” mechanism in plants. Rho GTPases act as molecular switches to regulate a variety of signal transduction pathways including cytoskeletal organization. Pathogens can modulate Rho GTPase activity to suppress host immune responses such as phagocytosis. Pyrin is encoded by MEFV, the gene that is mutated in patients with familial Mediterranean fever (FMF). FMF is the prototypic autoinflammatory disease characterized by recurring short episodes of systemic inflammation and is a common disorder in many populations in the Mediterranean basin. Pyrin specifically senses modifications in the activity of the small GTPase RhoA, which binds to many effector proteins including the serine/threonine-protein kinases PKN1 and PKN2 and actin-binding proteins. RhoA activation leads to PKN-mediated phosphorylation-dependent pyrin inhibition. Conversely, pathogen virulence factors downregulate RhoA activity in a variety of ways, and these changes are detected by the pyrin inflammasome irrespective of the type of modifications. MEFV pathogenic variants favor the active state of pyrin and elicit proinflammatory cytokine release and pyroptosis. They can be inherited either as a dominant or recessive trait depending on the variant's location and effect on the protein function. Mutations in the C-terminal B30.2 domain are usually considered recessive, although heterozygotes may manifest a biochemical or even a clinical phenotype. These variants are hypomorphic in regard to their effect on intramolecular interactions, but ultimately accentuate pyrin activity. Heterozygous mutations in other domains of pyrin affect residues critical for inhibition or protein oligomerization, and lead to constitutively active inflammasome. In healthy carriers of FMF mutations who have the subclinical inflammatory phenotype, the increased activity of pyrin might have been protective against endemic infections over human history. This finding is supported by the observation of high carrier frequencies of FMF-mutations in multiple populations. The pyrin inflammasome also plays a role in mediating inflammation in other autoinflammatory diseases linked to dysregulation in the actin polymerization pathway. Therefore, the assembly of the pyrin inflammasome is initiated in response to fluctuations in cytoplasmic homeostasis and perturbations in cytoskeletal dynamics.

The pyrin inflammasome has evolved as an innate immune sensor to detect bacterial toxin-induced Rho guanosine triphosphatase (Rho GTPase)-inactivation, a process that is similar to the "guard" mechanism in plants. Rho GTPases act as molecular switches to regulate a variety of signal transduction pathways including cytoskeletal organization. Pathogens can modulate Rho GTPase activity to suppress host immune responses such as phagocytosis. Pyrin is encoded by MEFV , the gene that is mutated in patients with familial Mediterranean fever (FMF). FMF is the prototypic autoinflammatory disease characterized by recurring short episodes of systemic inflammation and is a common disorder in many populations in the Mediterranean basin. Pyrin specifically senses modifications in the activity of the small GTPase RhoA, which binds to many effector proteins including the serine/threonine-protein kinases PKN1 and PKN2 and actin-binding proteins. RhoA activation leads to PKN-mediated phosphorylation-dependent pyrin inhibition. Conversely, pathogen virulence factors downregulate RhoA activity in a variety of ways, and these changes are detected by the pyrin inflammasome irrespective of the type of modifications. MEFV pathogenic variants favor the active state of pyrin and elicit proinflammatory cytokine release and pyroptosis. They can be inherited either as a dominant or recessive trait depending on the variant's location and effect on the protein function. Mutations in the C-terminal B30.2 domain are usually considered recessive, although heterozygotes may manifest a biochemical or even a clinical phenotype. These variants are hypomorphic in regard to their effect on intramolecular interactions, but ultimately accentuate pyrin activity. Heterozygous mutations in other domains of pyrin affect residues critical for inhibition or protein oligomerization, and lead to constitutively active inflammasome. In healthy carriers of FMF mutations who have the subclinical inflammatory phenotype, the increased activity of pyrin might have been protective against endemic infections over human history. This finding is supported by the observation of high carrier frequencies of FMF-mutations in multiple populations. The pyrin inflammasome also plays a role in mediating inflammation in other autoinflammatory diseases linked to dysregulation in the actin polymerization pathway. Therefore, the assembly of the pyrin inflammasome is initiated in response to fluctuations in cytoplasmic homeostasis and perturbations in cytoskeletal dynamics. [ABSTRACT FROM AUTHOR]

Pre‐axial polydactyly (PPD) is characterized by well‐developed non‐functional 1st digit (thumb) duplication in hands and/or feet. It is mostly inherited in autosomal dominant manner. In the present study, two families of Pakistani origin, demonstrating unilateral PPD type A, have been characterized at clinical and genetic levels. Whole‐exome sequencing (WES) revealed a nonsense mutation (c.84C > A, p.Tyr28*) in the STKLD1, located on chromosome 9q34.2, in affected individuals of both the families. Our findings report the first direct involvement of the STKLD1 in the digit development and highlight the importance of inclusion of this gene for screening individuals presenting non‐syndromic recessive PPD. [ABSTRACT FROM AUTHOR]

Improper signaling of the nuclear factor-κB (NF-κB) pathway plays a critical role in many inflammatory disease states including cancer, stroke, and viral infections. Although the signaling pathways are known, how these molecular mechanisms respond to changes in the intracellular microenvironment such as pH, ionic strength, and temperature, remains elusive. Molecular dynamics simulations were employed to differentiate the structural dynamics of the NF-κB Inducing Kinase (NIK), a protein kinase responsible for invoking the non-canonical NF-κB pathway, in its native and mutant form, and in the absence and presence of salt concentration in efforts to probe whether changes in the ionic environment stabilize or destabilize the NIK dimer. Analyses of structure–activity and conformational-activity relationships indicate that the protein–protein interactions are sensitive to changes in the ionic strength. Ligand binding pockets as well as regions between the oligomer interface either compress or expand, affecting both local and distal intermolecular interactions that result in stabilization or destabilization in the protein assembly. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor associated kinase 4 (IRAK4) has been implicated in IL-1R and TLR based signaling. Therefore selective inhibition of the kinase activity of this protein represents an attractive target for the treatment of inflammatory diseases. Medicinal chemistry optimization of high throughput screening (HTS) hits with the help of structure based drug design led to the identification of orally-bioavailable quinazoline based IRAK4 inhibitors with excellent pharmacokinetic profile and kinase selectivity. These highly selective IRAK4 compounds show activity in vivo via oral dosing in a TLR7 driven model of inflammation. [ABSTRACT FROM AUTHOR]

The gene PINK1 [phosphatase and tensin homologue (PTEN)-induced putative kinase 1] encodes a serine/threonine kinase which was initially linked to the pathogenesis of a familial form of Parkinson's disease. Research on PINK1 has recently unravelled that its multiple functions extend well beyond neuroprotection, implicating this eclectic protein in a growing number of human pathologies, including cancer, diabetes, cardiopulmonary dysfunctions, and inflammation. Extensive studies have identified PINK1 as a crucial player in the mitochondrial quality control pathway, required to label damaged mitochondria and promote their elimination through an autophagic process (mitophagy). Mounting evidence now indicates that PINK1 activities are not restricted solely to mitophagy, and that different subcellular and even sub-mitochondrial pools of PINK1 are involved in distinct signalling cascades to regulate cell metabolism and survival. In this review, we provide a concise overview on the different functions of PINK1 and their potential role in human diseases. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress.

A major goal of plant genome research is to recognize genes responsible for important traits. Resistance genes are among the most important gene classes for plant breeding purposes being responsible for the specific immune response including pathogen recognition, and activation of plant defence mechanisms. These genes are quite abundant in higher plants, with 210 clusters found in Eucalyptus FOREST database presenting significant homology to known R-genes. All five gene classes of R-genes with their respective conserved domains are present and expressed in Eucalyptus. Most clusters identified (93) belong to the LRR-NBS-TIR (genes with three domains: Leucine-rich-repeat, Nucleotide-binding-site and Toll interleucine 1-receptor), followed by the serine-threonine-kinase class (49 clusters). Some new combinations of domains and motifs of R-genes may be present in Eucalyptus and could represent novel gene structures. Most alignments occurred with dicots (94.3%), with emphasis on Arabidopsis thaliana (Brassicaceae) sequences. All best alignments with monocots (5.2%) occurred with rice (Oryza sativa) sequences and a single cluster aligned with the gymnosperm Pinus sylvestris (0.5%). The results are discussed and compared with available data from other crops and may bring useful evidences for the understanding of defense mechanisms in Eucalyptus and other crop species.

IRAK4 plays a critical role in the IL-1R and TLR signalling, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of permeable N -(1 H -pyrazol-4-yl)carboxamides was developed by introducing lipophilic bicyclic cores in place of the polar pyrazolopyrimidine core of 5-amino- N -(1 H -pyrazol-4-yl)pyrazolo[1,5- a ]pyrimidine-3-carboxamides. Replacement of the pyrazolo[1,5- a ]pyrimidine core with the pyrrolo[2,1- f ][1,2,4]triazine, the pyrrolo[1,2- b ]pyridazine, and thieno[2,3- b ]pyrazine cores guided by c Log D led to the identification of highly permeable IRAK4 inhibitors with excellent potency and kinase selectivity. [ABSTRACT FROM AUTHOR]

Objective To investigate the expressions of serine-threonine kinase (Akt)/ mammalian target of rapamycin (mTOR)/p?0 S6K in oral squamous cell carcinoma (OSCC) and provide references for early diagnosis and prognosis evaluation of OSCC. Methods A total of 51 cases of OSCC, 10 cases of paracancerous mucosa, and 10 cases of normal oral mucosa were collected. The expressions of Akt/mTOR/p70 S6K in these cases were detected using the SP method of immunohistochemistry. The correlation between their expressions in OSCC was also analyzed. Results The positive expressions of p-Akt, p-mTOR, and p70 S6K in OSCC were significantly higher than those in normal oral mucosa and paracancerous mucosa. The expressions of p-Akt, p-mTOR, and p70 S6K in OSCC were not correlated with age, gender, and clinical stage; by comparison, these expressions were correlated with lymph node metastasis and pathological grade. Strong positive correlations were also observed between the expressions of p-Akt, p-mTOR, and p70 S6K in OSCC. Conclusion Akt/mTOR/p70 S6K signaling molecules exhibit active expressions in OSCC and may be implicated in the occurrence and development of OSCC. [ABSTRACT FROM AUTHOR]

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a short time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococcus salivarius. We show that initiation of competence increases upon cell wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCE Survival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

Cytosolic PIM kinases are the members of serine/ threonine family play a crucial role in the cancer progression and development. Overexpression of PIM kinases is observed in various types of cancers including prostate, hematological, pancreatic, breast carcinoma and likewise. PIM kinases have now been considered as limelight target for the discovery of new molecules as novel anticancer agents as no drug is in the market targeting PIM kinases. In the last two decades, numerous PIM kinase inhibitors have been developed and few of them were in clinical trial phases but could not pass the pipeline of the clinical trials. The present comprehensive review intends to cover biological and the structural aspects of PIM kinases and also medicinal chemistry of PIM inhibitors developed in recent years. • PIM kinases considered as a new target for the discovery of new drug candidates against prostate and hematological cancers. • Structural insights of the PIM kinase X-Ray crystal structure. • Strategies to develop PIM kinase inhibitors based on the crystal structure of PIM kinases. • Detailed report of the PIM kinases inhibitors reported since two decades (2000–2021). • Mutations in the PIM kinase. [ABSTRACT FROM AUTHOR]

Abstract: Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A. The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3. [Copyright &y& Elsevier]

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) exhibit broad spectra of biological activities in various tissues, including bone, cartilage, blood vessels, heart, kidney, neurons, liver and lung. BMPs are members of the transforming growth factor-β (TGF-β) family that bind to type II and type I serine-threonine kinase receptors, and transduce signals through Smad and non-Smad signalling pathways. Recent findings have revealed that BMP signalling is finely tuned by various mechanisms in both positive and negative fashions. Perturbations of BMP signalling pathways are linked to a wide variety of clinical disorders, including vascular diseases, skeletal diseases and cancer. Administration of recombinant BMP ligands and increasing endogenous expression of BMPs provide therapeutic effects on some diseases. The recent development of BMP receptor inhibitors may also prove useful for some clinical diseases induced by hyperactivation of the BMP signalling pathways. [ABSTRACT FROM PUBLISHER]

Degenerate oligonucleotide primers, designed based on conserved regions of several serine-threonine kinases (STK) previously cloned in tomato and Arabidopsis, were used to isolate STK candidates in wild and cultivated strawberries. Seven distinct classes of STKs were identified from three related wild species, i.e., Fragaria vesca, Fragaria chiloensis, and Potentilla tucumanensis, and seven different Fragaria × ananassa cultivars. Alignment of the deduced amino acid sequences and the Pto R protein from tomato revealed the presence of characteristic subdomains and conservation of the plant STK consensus and other residues that are crucial for Pto function. Based on identity scores and clustering in phylogenetic trees, five groups were recognized as Pto-like kinases. Strawberry Pto-like clones presented sequences that were clearly identified as the activation segments contained in the Pto, and some of them showed residues previously identified as being required for binding to AvrPto. Some of the non-Pto-like kinases presented a high degree of identity and grouped together with B-lectin receptor kinases that are also involved in disease resistance. Statistical studies carried out to evaluate departure from the neutral theory and nonsynonymous/synonymous substitutions suggest that the evolution of STK-encoding sequences in strawberries is subjected mainly to a purifying selection process. These results represent the first report of Pto-like STKs in strawberry. [ABSTRACT FROM AUTHOR]

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most devastating diseases in rice worldwide. The dominant resistance gene, Pi-d2 [previously named Pi-d(t)2], present in the rice variety Digu, confers gene-for-gene resistance to the Chinese blast strain, ZB15. Pi-d2 was previously mapped close to the centromere of chromosome 6. In this study, the Pi-d2 gene was isolated by a map-based cloning strategy. Pi-d2 encodes a receptor-like kinase protein with a predicted extracellular domain of a bulb-type mannose specific binding lectin (B-lectin) and an intracellular serine–threonine kinase domain. Pi-d2 is a single-copy gene that is constitutively expressed in the rice variety Digu. Transgenic plants carrying the Pi-d2 transgene confer race-specific resistance to the M. grisea strain, ZB15. The Pi-d2 protein is plasma membrane localized. A single amino acid difference at position 441 of Pi-d2 distinguishes resistant and susceptible alleles of rice blast resistance gene Pi-d2. Because of its novel extracellular domain, Pi-d2 represents a new class of plant resistance genes. [ABSTRACT FROM AUTHOR]

The family of testis-specific serine–threonine kinases (TSSKs) consists of four members whose expression is confined almost exclusively to testis. Very little is known about their physiological role and mechanisms of action. We cloned human and mouse TSSK3 and analysed the biochemical properties, substrate specificity and in vitro activation. In vitro TSSK3 exhibited the ability to autophosphorylate and to phosphorylate test substrates such as histones, myelin basic protein and casein. Interestingly, TSSK3 showed maximal in vitro kinase activity at 30 °C, in keeping with it being testis specific. Sequence comparison indicated the existence of a so-called ‘T-loop’ within the TSSK3 catalytic domain, a structure present in the AGC family of protein kinases. To test if this T-loop is engaged in TSSK3 regulation, we mutated the critical threonine residue within the T-loop to alanine (T168A) which resulted in inactivation of TSSK3 kinase. Furthermore, Thr168 is phosphorylated in vitro by the T-loop kinase phosphoinositide-dependent protein kinase-1 (PDK1). PDK1-induced phosphorylation increased in vitro TSSK3 kinase activity, suggesting that TSSK3 can be regulated in the same way as AGC kinase family members. Analysis of peptide sequences identifies the peptide sequence RRSSSY containing Ser5 that is a target for TSSK3 phosphorylation, as an efficient and specific substrate for TSSK3. [ABSTRACT FROM AUTHOR]

HIPK2 is a member of a novel family of nuclear serine–threonine kinases identified through their ability to interact with the Nkx-1.2 homeoprotein. The physiological role of these kinases is largely unknown, but we have recently reported on the involvement of HIPK2 in the induction of apoptosis of tumor cells after UV stress through p53 phosphorylation and transcriptional activation. Here, we demonstrate that the chemotherapeutic drug cisplatin increases HIPK2 protein expression and its kinase activity, and that HIPK2 is involved in cisplatin-dependent apoptosis. Indeed, induction of HIPK2 and of cell death by cisplatin are efficiently inhibited by the serine–threonine kinase inhibitor SB203580 or the transduction of HIPK2-specific RNA-interfering molecules. HIPK2 gene silencing efficiently reduces the p53-mediated transcriptional activation of apoptotic gene promoters as well as apoptotic cell death after treatment with cisplatin. These findings, along with the involvement of p53 phosphorylation at serine 46 (Ser46) in the transcriptional activation of apoptotic gene promoters, suggest a critical role for HIPK2 in triggering p53-dependent apoptosis in response to the antineoplastic drug cisplatin. [Copyright &y& Elsevier]

The p21-activated kinase (PAK) family is homologous to the yeast sterile 20 (Ste20) and regulates a wide variety of cellular responses, including cell morphology, proliferation, and survival. In this study we examined the activation of PAK1 by Gβγ subunits. Co-transfection of COS7 cells with Gβ1γ2 or Gβ1γ5 was sufficient to induce agonist-independent activation of PAK1. Expression of dominant/negative Rac, Cdc42, or Ras did not inhibit this Gβγ-dependent activation. Wortmannin, which inhibits phosphoinositide 3-kinase (PI3-kinase) activity, and expression of a dominant/negative form of Akt were sufficient to abrogate the activation of PAK1 that was induced by Gβγ. These results reveal that stimulation of PAK1 by Gβγ can occur via a PI3-kinase and Akt pathway that does not require Rac1 or Cdc42. [Copyright &y& Elsevier]

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis. [Copyright &y& Elsevier]

Par-1 encodes a serine/threonine kinase that is involved in asymmetric segregation of cell fate determinants in Caenorhabditis elegans and Drosophila embryos. Recent biochemical studies indicate an association of PAR-1 with the Dishevelled protein and suggest a role in so-called canonical Wnt signaling (Nat. Cell Biol. 3 (2001) 628). Here we describe two Xenopus laevis cDNAs, which encode PAR-1 homologues designated XPar-1A and XPar-1B. Structurally, XPar-1A and XPar-1B are closely related to rat MARK proteins and human Par-1A and Par-1Bα, respectively. XPar-1A and XPar-1B are expressed both maternally and zygotically in an indistinguishable pattern. In the egg and cleavage stage embryos their transcripts are enriched in the animal pole of the embryo. During blastula and gastrula stages, cells in the animal and marginal regions continue to express both genes uniformly. Expression progresses vegetally towards and then through the blastopore lip concomitantly with the movements of epiboly and gastrulation. With the onset of neurulation, XPar-1A and XPar-1B transcripts are restricted to the neurectoderm. At tailbud and tadpole stages they are detected in the head region, including brain, eyes, otic vesicles, cement gland, branchial arches as well as spinal cord and somites. Therefore, this analysis suggests that the Xenopus par-1 homologues XPar-1A and XPar-1B are expressed in frog embryos both maternally and zygotically in a restricted pattern and may play a role in establishing polarity in early embryos as well as in organogenesis during later stages of development. [Copyright &y& Elsevier]

The vaccinia-related kinase 1 (VRK1) protein is a nuclear Ser-Thr kinase that phosphorylates p53 in Thr18. We have determined the enzyme properties regarding its different substrates. VRK1 has a high affinity for ATP (Km 50 μM) and is thus saturated by the intracellular concentration of ATP in vivo. VRK1 uses preferentially magnesium, but is also functional with manganese and zinc. The VRK1 protein is autophosphorylated in multiple residues without effect on its activity. One autophosphorylated residue, T355, is within the VRK1 regulatory carboxy terminus. The kinase phosphorylates p53 with a Km of 1 μM and is well suited to respond to the variations of intracellular p53 concentration, which fluctuates as a response to different types of cellular stress. [Copyright &y& Elsevier]

The HIPK2 protein is a member of a recently identified family of nuclear protein kinases that interact with homeodomain transcription factors and that are well conserved in various organisms. HIPK2 acts as a transcriptional co-repressor of homeoproteins and it is a component of a co-repressor complex containing Groucho and a histone deacetylase. We have recently demonstrated that the HIPK2 protein interacts with HMGA1 proteins in vitro and in vivo and that HIPK2 exerts a potent inhibitory effect on the cell growth of different normal cell lines. Here we demonstrate that the HIPK2 gene is expressed during the latest phases of embryogenesis, and preferentially in retina, in muscle and in neural tissues. Conversely, HIPK2 is ubiquitously expressed in murine and human adult tissues. We have also found that HIPK2 expression is reduced in breast and thyroid carcinomas, suggesting a role of this gene in the process of carcinogenesis. [Copyright &y& Elsevier]

Background: The novel serine-threonine kinase Akt is a critical enzyme in cell survival. We investigated the roles of the Akt pathway and apoptotic signals in (1) Madin-Darby canine kidney (MDCK) cells in a hyperosmotic condition in vitro and (2) in the inner medulla of dehydrated rat in vivo.Methods: The in vivo experiments were performed in 24- and 48-hour water-restricted rats. Hyperosmolality-stimulated Akt phosphorylation was examined in MDCK cells. Phosphatidylinositol 3-kinase (PI3-K) inhibitors, the dominant-negative mutant of PI3-K, the dominant-negative mutant of Akt, and the dominant-active form of Akt were used to examine the roles of the PI3-K/Akt pathways in renal tubular cell apoptosis.Results: The amount of phosphorylated Akt protein was increased in the inner medulla of dehydrated rats. Hyperosmolality induced by the addition of NaCl, urea, and raffinose phosphorylated Akt in MDCK cells in an osmolality-dependent manner. PI3-K inhibitors and the dominant-negative mutant of PI3-K inhibited the hyperosmolality-induced phosphorylation of Akt. Raising the media osmolality from a normal level to 500 or 600 mOsm/kg H2O final osmolality elicited apoptotic changes such as nucleosomal laddering of DNA and an increment of caspase-3 activity and increased activity in the cell death enzyme-linked immunosorbent assay. Dominant-active Akt prevented the mild hyperosmolality-induced apoptosis, while inhibition of the PI3-K/Akt pathways promoted apoptosis.Conclusion: The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis. [ABSTRACT FROM AUTHOR]

Role of SGK in mineralocorticoid-regulated sodium transport. Mineralocorticoids stimulate electrogenic Na+ transport in tight epithelia by altering the transcription of specific genes. Although the earliest mineralocorticoid effect is to increase the activity of the epithelial sodium channel (ENaC), ENaC mRNA and protein levels do not change. Instead, physiologic observations suggest that a mineralocorticoid target gene(s) encodes an ENaC regulator(s). To begin to identify and characterize mineralocorticoid-regulated target genes, we used suppression-subtractive hybridization to generate a cDNA library from A6 cells, a stable cell line of Xenopus laevis of distal nephron origin. A serine-threonine kinase, SGK, was identified from this screen. Sequence comparison revealed that frog, rat, and human SGK are 92% identical and 96% similar at the amino acid level. SGK mRNA was confirmed by Northern blot to be strongly and rapidly corticosteroid stimulated in A6 cells. In situ hybridization revealed that SGK was strongly stimulated by aldosterone in rat collecting duct but not proximal tubule cells. Low levels of SGK were present in rat glomeruli, but SGK was unregulated in this structure. Finally, SGK stimulated ENaC activity approximately sevenfold when coexpressed in Xenopus laevis oocytes. These data suggest that SGK is an important mediator of aldosterone effects on Na+ transport in tight epithelia. In view of the existence of SGK homologues in invertebrates, it is interesting to speculate that SGK is an ancient kinase that was adapted to the control of epithelial Na+ transport by early vertebrates as they made the transition from a marine to a freshwater environment. [ABSTRACT FROM AUTHOR]

We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Star 5B and the receptor requires a functional insulin-receptor kinase, Tyr960 of insulin receptor is implicated in the interaction with Star 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated tit Thr684, a potential phosphorylation site of mitogen-activated protein kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells. Taken together our findings suggest that Stat 5B corresponds to a substrate for the insulin-receptor kinase, and this widens the repertoire of insulin-signaling pathways. [ABSTRACT FROM AUTHOR]

Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases. Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies.We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae Cdc7 kinase is essential for initiation of DNA replication, and Hsk1, a related kinase of Schizosaccharomyces pombe, is also required for DNA replication of fission yeast cells. We report here cDNAs encoding Cdc7-related kinases from human and Xenopus (huCdc7 and xeCdc7, respectively). The cloned cDNA for huCdc7 contains an open reading frame consisting of 574 amino acids with a predicted molecular weight of 63 847 that possesses overall amino acid identity of 32% (54% including similar residues) to Cdc7 and Hsk1. huCDC7 is transcribed in the various tissues examined, but most abundantly in testis. Three transcripts of 4.4, 3.5 and 2.4 kb in length are detected. The 3.5 kb transcript is the most predominant and is expressed in all the tissues examined. A cDNA containing a 91 nucleotide insertion at the N-terminal region of huCDC7 is also detected, suggesting the presence of multiple splicing variants. The huCdc7 protein is expressed at a constant level during the mitotic cell cycle and is localized primarily in nuclei in interphase and distributed diffusibly in cytoplasm in the mitotic phase. The wild-type huCdc7 protein expressed in COS7 cells phosphorylates MCM2 and MCM3 proteins in vitro, suggesting that huCdc7 may regulate processes of DNA replication by modulating MCM functions. [ABSTRACT FROM AUTHOR]

The type II transforming growth factor-β (TGF-β) receptor Ser/Thr kinase (TβRII) is responsible for the initiation of multiple TGF-β signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TβRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation which is essential for the activation of TβRII kinase activity, activation of TβRI and TGF-β-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an inter-molecular mechanism. Phosphorylation of Ser409 is essential for TβRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TβRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TβRII phosphorylation is regulated intricately and affects TGF-β receptor signal transduction both positively and negatively. [ABSTRACT FROM AUTHOR]

DAP-kinase was initially identified as a gene whose anti-sense-mediated reduced expression protected HeLa cells from interferon-γ-induced programmed cell death. It was cloned in our laboratory by a functional gene selection approach. According to its amino acid sequence, this 160 kDa protein was predicted to be a novel type of calmodulin-regulated serine/threonine kinase which carries ankyrin repeats and the death domain. In this work we have shown that the kinase was autophosphorylated and capable of phosphorylating an exogenous substrate in a Ca2+ /calmodulin-dependent manner. We proved that calmodulin binds directly to the recombinant kinase, and generated a constitutively active kinase mutant by the deletion of the calmodulin regulatory domain. By immunostaining and biochemical fractionations we demonstrated that the kinase is localized to the cytoskeleton, in association with the microfilament system, and mapped a region within the protein which is responsible for binding to the cytoskeleton. Several assays attributed a cell death function to the gene. Ectopic expression of wild-type DAP-kinase induced the death of target cells, and the killing property depended strictly on the status of the intrinsic kinase activity. Conversely, a catalytically inactive mutant that carried a lysine to alanine substitution within the kinase domain, displayed dominant-negative features and protected cells from interferon-γ-induced cell death. DAP-kinase is therefore a novel cytoskeletal-associated cell death serine/threonine kinase whose activation by Ca2+ /calmodulin may be linked to the biochemical mechanism underlying the cytoskeletal alterations that occur during cell death. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient's tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies. [ABSTRACT FROM AUTHOR]

Leucine-rich repeat kinase 2 (LRRK2) is a serine-threonine kinase involved in multiple cellular processes and signaling pathways. LRRK2 mutations are associated with autosomal-inherited Parkinson's disease (PD), and evidence suggests that LRRK2 pathogenic variants generally increase kinase activity. Therefore, inhibition of LRRK2 kinase function is a promising therapeutic strategy for PD treatment. The search for drug-like molecules capable of reducing LRRK2 kinase activity in PD led to the design of selective LRRK2 inhibitors predicted to be within the CNS drug-like space. This review highlights the journey that translates chemical tools for interrogating the role of LRRK2 in PD into promising drug candidates, addressing the challenges in discovering selective and brain-penetrant LRRK2 modulators and exploring the structure-activity relationship of distinct LRRK2 inhibitors.