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Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCa and a novel role for PKCa in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)+ binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCa. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCa-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCa and G3BP2 indicated that the interaction is direct and PKCa can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCa and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2a are suppressed following downregulation of PKCa by siRNA. In conclusion this study identifies novel interaction partners of PKCa and a novel role for PKCa in regulation of stress granules. [ABSTRACT FROM AUTHOR]

Abstract: Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named Rmyt1 and Rmyt2, were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only Rmyt2 was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2α) itself was sufficient to phosphorylate Rmyt2, but phosphorylation was enhanced by the presence of the regulatory subunit CK2β. Even in the absence of CK2, Rmyt2 was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of Rmyt2 to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of Rmyt2 showed that mussel protein contains the signature sequence common to all PKA family members, within the “phosphate binding cassette” (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from Rmyt2, as a whole, and those from type II R subunits was 68–75%, but the global identity percentage with type I R subunits was only about 30%, so that Rmyt2 can be classified as a type II R subunit. [Copyright &y& Elsevier]

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling. J. Cell. Physiol. 206: 457–467, 2006. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Tyr-762 is an autophosphorylation site in the human platelet-derived growth factor (PDGF) α-receptor. In order to investigate whether phosphorylated Tyr-762 serves as a docking site for downstream signal transduction molecules, affinity purification using an immobilized synthetic peptide containing phosphorylated Tyr-762 and its surrounding amino acid residues was performed. Proteins in HeLa cell lysate of molecular sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to the unphosphorylated peptide. Analyses of partial amino acid sequences of the purified proteins indicated that they were identical to CrkI, CrkII and CrkL respectively. The wild-type PDGF α-receptor, when expressed in porcine aortic endothelial cells, formed complexes with CrkII and CrkL upon ligand stimulation, which was specifically inhibited by a synthetic peptide containing phosphorylated Tyr-762. Replacement of Tyr-762 with a phenylalanine residue in the PDGF α-receptor abrogated ligand-induced binding of Crk proteins. Tyrosine phosphorylation of CrkII and CrkL increased by 1.8- and 1.3-fold, respectively, upon ligand stimulation of the wild-type α-receptor. In contrast, the Y762F mutant PDGF α-receptor failed to induce tyrosine phosphorylation of Crk proteins. CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well as PDGF-stimulated cells. Moreover, the activated wild-type PDGF α-receptor but not the Y762F mutant receptor was found in a C3G immunoprecipitate, suggesting that a ternary complex between the activated PDGF α-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in cells expressing wild-type and mutant receptors. Interestingly, the activated PDGF β-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the β-receptor, which is localized at an analogous position to Tyr-762 in the... [ABSTRACT FROM AUTHOR]

Abstract: Thymidylate synthase (TS) was found to be a substrate for both catalytic subunits of human CK2, with phosphorylation by CK2α and CK2α′ characterized by similar Km values, 4.6μM and 4.2μM, respectively, but different efficiencies, the apparent turnover number with CK2α being 10-fold higher. With both catalytic subunits, phosphorylation of human TS, like calmodulin and BID, was strongly inhibited in the presence of the regulatory subunit CK2β, the holoenzyme being activated by polylysine. Phosphorylation of recombinant human, rat, mouse and Trichinella spiralis TSs proteins was compared, with the human enzyme being apparently a much better substrate than the others. Following hydrolysis and TLC, phosphoserine was detected in human and rat, and phosphotyrosine in T. spiralis, TS, used as substrates for CK2α. MALDI-TOF MS analysis led to identification of phosphorylated Ser124 in human TS, within a sequence LGFS124TREEGD, atypical for a CK2 substrate recognition site. The phosphorylation site is located in a region considered important for the catalytic mechanism or regulation of human TS, corresponding to the loop 107–128. Following phosphorylation by CK2α, resulting in incorporation of 0.4mol of phosphate per mol of dimeric TS, human TS exhibits unaltered Km values for dUMP and N5,10-methylenetetrahydrofolate, but a 50% lower turnover number, pointing to a strong influence of Ser124 phosphorylation on its catalytic efficiency. [Copyright &y& Elsevier]

We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor. [Copyright &y& Elsevier]

Reversible phosphorylation of acidic ribosomal proteins of Saccharomyces cerevisiae is an important mechanism, regulating the number of active ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. A new protein-inhibitor regulating activity of PK60S kinase has been purified from yeast extracts and characterised. Peptide mass fingerprinting (PMF) and amino-acid sequence analysis by Post Source Decay (PSD) have identified the inhibitor as a Cu–Zn superoxide dismutase (SOD). Inhibition by SOD is competitive with respect to protein substrates—P proteins and 80S ribosome—with Ki values of 3.7 μM for P2A protein and 0.6 μM for 80S ribosomes. A close correlation was found between the state of phosphorylation of P proteins in diauxic shift and logarithmic growth yeast cells and activity of SOD. The possible mechanism of regulation of PK60S activity, and participation of SOD protein in regulation of 80S-ribosome activity in stress conditions, is discussed. [Copyright &y& Elsevier]

DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair. [ABSTRACT FROM AUTHOR]