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Serine/threonine Protein Kinase (threonine + protein_kinase)
Selected AbstractsCharacterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substratesFEBS JOURNAL, Issue 5 2005Linda Nováková Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP- N -acetylglucosamine, an essential common precursor to cell envelope components. [source] Protein phosphorylation pathways involved during lipopolysaccharide-induced expression of CD14 in mouse bone marrow granulocytesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2000Thierry Pedron Abstract Lipopolysaccharide (LPS) of Gram-negative bacteria interacts with a CD14-independent receptor of mouse bone marrow granulocytes (BMC), and triggers in these cells the expression of CD14, an inducible type of LPS receptor (iLpsR). This particular response of BMC to LPS required the activation of protein tyrosine kinase and p38 MAP kinase. The inhibition of the LPS effect by the MEK inhibitor PD-98059 suggested that the ERK pathway was also involved. Unexpectedly, protein kinase C, myosin light chain kinase, cAMP-, cGMP-, and Ca2+/calmodulin-dependent kinases, as well as ecto-protein kinases, were not required for iLpsR expression. However, other yet unidentified serine/threonine protein kinase(s) were implied since the BMC response to LPS was markedly reduced after exposure to three inhibitors of such kinases (K-252a, H-7, and KT-5823). The atypical kinase requirements observed in this study may be due either to a novel signaling LPS receptor complex present in BMC, or to the particular events involved in CD14 biosynthesis. [source] Different cellular localization, translocation, and insulin-induced phosphorylation of PKB, in HepG2 cells and hepatocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2002Noor Afshan Syed Abstract Protein kinase B (PKB), a serine/threonine protein kinase, prevents apoptosis and promotes cellular transformation. PKB activity is stimulated by insulin. In this report, we examined the relative amounts of expression, location, and translocation upon insulin stimulation of PKB, in normal primary hepatocytes and carcinoma cells, HepG2 cells. Non-phosphorylated PKB, was present in both types of unstimulated cells. The phosphorylated form of the enzyme was present in the nucleus of unstimulated HepG2 cells but not in normal hepatocytes. In the cytoplasm, PKB, was found in greater abundance in the hepatocytes as compared in HepG2 cells. Insulin induced the translocation of phosphorylated PKB, from the nucleus to the nuclear membrane in HepG2 cells. In contrast, insulin caused translocation and phosphorylation of PKB, from the cytosol to the plasma membrane in normal hepatocytes. In addition, there is a higher expression of PKB, in the HepG2 cells as compared to normal primary hepatocytes. These findings provide an important distinction between hepatocellular HepG2 cells and normal liver cells and suggest that the presence of constitutively active nuclear PKB in the transformed cells might be an important contributor in cell transformation and immortality of hepatoma cells. J. Cell. Biochem. 86: 118,127, 2002. © 2002 Wiley-Liss, Inc. [source] Noradrenaline enhances the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of PI3K/Akt and the mTOR/S6K pathwayJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Julie Chenal Abstract Monocarboxylate transporter 2 (MCT2) expression is up-regulated by noradrenaline (NA) in cultured cortical neurons via a putative but undetermined translational mechanism. Western blot analysis showed that p44/p42 mitogen-activated protein kinase (MAPK) was rapidly and strongly phosphorylated by NA treatment. NA also rapidly induced serine/threonine protein kinase from AKT virus (Akt) phosphorylation but to a lesser extent than p44/p42 MAPK. However, Akt activation persisted over a longer period. Similarly, NA induced a rapid and persistent phosphorylation of mammalian target of rapamycin (mTOR), a kinase implicated in the regulation of translation in the central nervous system. Consistent with activation of the mTOR/S6 kinase pathway, phosphorylation of the ribosomal S6 protein, a component of the translation machinery, could be observed upon treatment with NA. In parallel, it was found that the NA-induced increase in MCT2 protein was almost completely blocked by LY294002 (phosphoinositide 3-kinase inhibitor) as well as by rapamycin (mTOR inhibitor), while mitogen-activated protein kinase kinase and p38 MAPK inhibitors had much smaller effects. Taken together, these data reveal that NA induces an increase in neuronal MCT2 protein expression by a mechanism involving stimulation of phosphoinositide 3-kinase/Akt and translational activation via the mTOR/S6 kinase pathway. Moreover, considering the role of NA in synaptic plasticity, alterations in MCT2 expression as described in this study might represent an adaptation to face energy demands associated with enhanced synaptic transmission. [source] Target of rapamycin in the oriental fruit fly Bactrocera dorsalis (hendel): its cloning and effect on yolk protein expressionARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010R. Suganya Abstract Target of rapamycin (TOR), a serine/threonine protein kinase, is involved in regulating a number of growth and developmental processes of an organism, including yolk protein synthesis in insects. In this study, TOR gene was isolated, designated BdTOR (GenBank accession no. FJ167395), from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed a higher expression of BdTOR in the pupa than in other developmental stages, as well as in ovary than in the fat body. Downregulation of BdTOR activity by rapamycin treatment and RNA interference (RNAi) in vivo resulted in a significant reduction in yolk protein transcripts in both fat body and ovary, with a substantial reduction in ovary size. However, an unexpected increase in the expression of yolk protein gene was observed in adult ovary 9 days after rapamycin treatment. Taken together, the results suggest the involvement of BdTOR in the regulation of yolk protein synthesis in B. dorsalis. © 2010 Wiley Periodicals, Inc. [source] A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1CELLULAR MICROBIOLOGY, Issue 5 2006Andrea Haraga Summary Essential to salmonellae pathogenesis is an export device called the type III secretion system (TTSS), which mediates the transfer of bacterial effector proteins from the bacterial cell into the host cell cytoplasm. Once inside the host cell, these effectors are then capable of altering a variety of host cellular functions in order to promote bacterial survival and colonization. SspH1 is a Salmonella enterica serovar Typhimurium TTSS effector that localizes to the mammalian nucleus and down-modulates production of proinflammatory cytokines by inhibiting nuclear factor (NF)-,B-dependent gene expression. To identify mammalian binding partners of SspH1 a yeast two-hybrid screen against a human spleen cDNA library was performed. It yielded a serine/threonine protein kinase called protein kinase N 1 (PKN1). The leucine-rich repeat domain of SspH1 was demonstrated to mediate this interaction and also inhibition of NF-,B-dependent gene expression. This suggested that PKN1 may play a role in modulation of the NF-,B signalling pathway. Indeed, we found that expression of constitutively active PKN1 in mammalian cells results in a decrease, while depletion of PKN1 by RNA interference causes an increase in NF-,B-dependent reporter gene expression. These data indicate that SspH1 may inhibit the host's inflammatory response by interacting with PKN1. [source] A cyclic adenosine 3,,5,-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Hiroshi Harayama Abstract An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5°C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa. Mol. Reprod. Dev. 1169,1178, 2006. © 2006 Wiley-Liss, Inc. [source] The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: Mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175cPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2008Marc J. Canova Abstract Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis. [source] Structural insight into nucleotide recognition by human death-associated protein kinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Laurie K. McNamara Death-associated protein kinase (DAPK) is a member of the Ca2+/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg2+ at 1.85,Ĺ resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK,ADP,Mg2+ was compared with a newly determined DAPK,AMP-PNP,Mg2+ structure and the previously determined apo DAPK structure (PDB code 1jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK. [source] | |||||||||||||