Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.
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PMID:Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP. 222 78

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.
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PMID:A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation. 254 69

Eukaryotic initiation factor-2 (eIF-2) from Artemia embryos is able to exchange guanine nucleotides at the same rate in the presence or absence of Mg2+ when the reaction is carried out with either purified eIF-2 at 30 degrees C or less purified preparations at any temperature (10-30 degrees C). No exchange factor appears to catalyze this reaction. However, with purified eIF-2 at lower temperatures (10 degrees C) the exchange is clearly impaired by Mg2+ and this impairment is overcome by the guanine nucleotide exchange factor (GEF) of rabbit reticulocytes. Thus, Artemia eIF-2 is able to exchange guanine nucleotides by two alternative mechanisms that may reflect two states of the protein. Phosphorylation of the eIF-2 alpha subunit by the heme-controlled inhibitor (HCI) of rabbit reticulocytes abolishes the GEF-dependent reaction, but has no effect on the factor-independent one. The search for eIF-2 alpha kinases in Artemia embryo led to the detection of only one such enzyme, which was identified as a casein kinase type II. None of the exchange reactions is affected by the phosphorylation of the eIF-2 alpha subunit by this kinase, suggesting that, irrespective of the kind of mechanism for guanine nucleotide exchange that is actually operating in Artemia, it might not be a target for regulation by eIF-2 alpha phosphorylation.
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PMID:Phosphorylation and guanine nucleotide exchange on polypeptide chain initiation factor-2 from Artemia embryos. 290 42

T-cell antigen receptor (TCR) ligation of an Lck-deficient Jurkat mutant, J.CaM1, with anti-CD3 or anti-TCR beta monoclonal antibodies failed to induce tyrosine phosphorylation and activation of p42MAPK. The same stimuli activated mitogen-activated protein (MAP) kinase in J.CaM1 cells transfected with Lck, demonstrating that Lck plays a critical role in MAP kinase activation. Utilizing immunocomplex kinase assays, we demonstrated that TCR/CD3 ligation activated a MAP kinase kinase kinase (Raf-1) as well as a MAP kinase kinase (MEK-1) in Jurkat but not in J.CaM1 cells. It was possible, however, to activate Raf-1, MEK-1, and p42MAPK in J.CaM1 cells during treatment with the phorbol ester phorbol 12-myristate 13-acetate, which activates protein kinase C (PKC). This demonstrates the presence of a PKC-dependent pathway which functions independently from Lck in MAP kinase activation. Stimulation of Jurkat cells with either anti-TCR beta or anti-CD3 monoclonal antibody failed to induce substantial tyrosine phosphorylation of Shc proteins or their association with Grb2 which forms a complex with the guanine nucleotide exchange factor hSOS. However, the same stimuli induced tyrosine phosphorylation of another putative guanine nucleotide exchange factor, p95Vav, in Jurkat but not J.CaM1 cells. Moreover, Lck was reversibly co-immunoprecipitated with p95Vav, and the stoichiometry of binding increased in anti-CD3-treated Jurkat cells. Phorbol 12-myristate 13-acetate did not induce tyrosine phosphorylation of p95Vav. These data show that the TCR activates MAP kinase by way of a signaling cascade, which depends upon Lck, and may be mediated by downstream events involving PKC or p95Vav which act on Raf-1 and MEK-1.
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PMID:The T-cell antigen receptor utilizes Lck, Raf-1, and MEK-1 for activating mitogen-activated protein kinase. Evidence for the existence of a second protein kinase C-dependent pathway in an Lck-negative Jurkat cell mutant. 751 37

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.
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PMID:Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides. 751 72

In mammalian cells, the guanine nucleotide exchange factor (GEF, eIF-2B) plays a major role in the regulation of initiation of protein synthesis. It catalyzes the exchange of eukaryotic chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the recycling of eIF-2 during polypeptide chain initiation. We used the Friend virus-transformed murine erythroleukemia (MEL) cell system to elucidate the translational regulatory processes that occur during growth and hexamethylene bisacetamide (HMBA)-induced cell differentiation. GEF activity is increased during growth and decreased during MEL cell differentiation, and this parallels the overall changes in protein synthesis during this period. Inhibition of GEF activity in induced cells may occur indirectly by phosphorylation of the alpha-subunit of eIF-2. However, the decrease in GEF activity in induced cells cannot be reversed by increasing the concentration of eIF-2-GDP added as a substrate in the GEF assay. This is diagnostic for the presence of eIF-2 alpha(P)-GDP in cell lysates and suggests that regulation of GEF activity may occur by one or more mechanisms other than eIF-2(alpha) phosphorylation. We have previously shown that the activity of GEF may be influenced directly by phosphorylation with casein kinase II (CK-II) of the 82-kD subunit of the factor. CK-II activity parallels the changes in GEF activity and the rate of protein synthesis during growth and differentiation of MEL cells. Addition of 1mM spermidine, a stimulator of CK-II but not of purified GEF, in induced MEL cell extracts enhances both CK-II and GEF activities approximately 48 and 32%, respectively. The results presented suggest that the inhibition of protein synthesis during MEL cell differentiation may be linked to the decreased CK-II and GEF activities.
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PMID:Hexamethylene bisacetamide-induced differentiation of Friend virus-transformed murine erythroleukemia cells is associated with parallel changes in casein kinase II and guanine nucleotide exchange factor activities. 755 31

p21ras is a small GTPase that functions as a molecular switch in intracellular signal transduction pathways activated by a large variety of growth factors. In 50% of colorectal tumours, one of the genes for p21ras is mutated resulting in a constitutive active protein. Recently, progress has been made in the elucidation of the signalling pathways in which p21ras is involved. After a ligand binds to growth factor receptors, in particular receptor tyrosine kinases, a guanine nucleotide exchange factor is activated, which results in p21ras in the GTP-bound form. This GTP-bound form of p21ras interacts with the protein kinase raf1 and induces the activation of a kinase cascade, resulting in various cellular responses. This kinase cascade is part of an integrated network of both positive and negative signalling events.
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PMID:p21ras: an oncoprotein functioning in growth factor-induced signal transduction. 757 90

In mammalian cells, the guanine nucleotide exchange factor (GEF or eIF-2B) is a key regulator of polypeptide chain initiation. The exchange of GDP bound to chain initiation factor 2 (eIF-2) for GTP by GEF is a rate limiting step in protein synthesis. The multisubunit characteristics of GEF suggest that this protein is composed of several distinct structural and functional domains, and is regulated by allosteric means and by phosphorylation. The activity of GEF may be regulated indirectly by the phosphorylation state of the smallest subunit of eIF-2 (alpha-subunit). On the other hand, phosphorylation of the largest subunit of GEF (82-kD subunit) by casein kinase (CK) I or II stimulates GDP/GTP exchange. GEF contains NADPH which is required for structural integrity of the protein. Upon stimulation of cells by insulin and growth factors, allosteric activation of GEF by sugar phosphates and other effector molecules may also play an important role in the regulation of polypeptide chain initiation. In this article, recent information about structure-function relationship of eIF-2 and GEF in nucleotide exchange and the regulatory mechanisms that influence the rate of polypeptide chain initiation under various physiological and pathological conditions are presented.
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PMID:Translational control of eukaryotic gene expression. Role of the guanine nucleotide exchange factor and chain initiation factor-2. 758 45

Mitogen-activated protein (MAP) kinases p42mapk and p44mapk are activated in cells stimulated with epidermal growth factor (EGF) and other agents. A principal pathway for MAP kinase (MAPK) activation by EGF consists of sequential activations of the guanine nucleotide exchange factor Sos, the guanosine triphosphate binding protein Ras, and the protein kinases Raf-1, MAPK kinase (MKK), and MAPK. Because adenosine 3',5'-monophosphate (cAMP) does not activate MAPK and has some opposing physiologic effects, the effect of increasing intracellular concentrations of cAMP with forskolin and 3-isobutyl-1-methylxanthine on the EGF-stimulated MAPK pathway was studied. Increased concentrations of cAMP blocked activation of Raf-1, MKK, and MAPK in Rat1hER fibroblasts, accompanied by a threefold increase in Raf-1 phosphorylation on serine 43 in the regulatory domain. Phosphorylation of Raf-1 in vitro and in vivo reduces the apparent affinity with which it binds to Ras and may contribute to the blockade by cAMP.
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PMID:Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. 825 59

Stimulation of the prolactin receptor (PRLr) with ligand activates multiple kinase cascades. The proximal mediators involved in the activation of the PRL-activated Raf-1 cascade in T-cells, however, remain poorly characterized. The role of one proximal signaling protein, namely p95vav, during PRLr signal transduction was examined in the Nb2 T-cell line. The novel results obtained here indicate that Vav is transiently associated with the PRLr and is necessary for PRL-stimulated proliferation. During PRL stimulation, a rapid and dramatic increase in guanine nucleotide exchange factor (GEF) activity was found to be associated with Vav immunoprecipitates. Concomitantly, an increase in Vav phosphorylation on serine-threonine residues was observed. The Vav-associated GEF activation could be inhibited by staurosporine and calphostin, but not herbimycin, suggesting a modulatory role for phosphorylation at serine-threonine residues. Treatment of Nb2 cells with antisense Vav oligonucleotide ablated Vav expression and blocked PRL-driven proliferation, but failed to inhibit PRL-induced GEF activation within Nb2 lysates. These data indicate that GEF activity may not be intrinsic to Vav as has been previously suggested, but either resides in or is complemented by an associated GEF. Subsequent to the transient activation of associated GEF activity, Vav was found to translocate into the Nb2 cell nucleus. Thus, Vav may utilize two independent mechanisms in T-cells, namely the activation of an associated GEF and direct nuclear internalization, to mediate PRLr signaling.
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PMID:Vav is necessary for prolactin-stimulated proliferation and is translocated into the nucleus of a T-cell line. 776 23


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