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Query: EC:2.7.10.1 (ERK)
 95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used a genomic library of Candida albicans to transform Saccharomyces cerevisiae and screened for genes that act similarly to dominant negative mutations by interfering with pheromone-mediated cell cycle arrest. Six different plasmids were identified from 2000 transformants; four have been sequenced. One gene (CZF1) encodes a protein with structural motifs characteristic of a transcription factor. A second gene (CCN1) encodes a cyclin homologue, a third (CRL1) encodes a protein with sequence similarity to GTP-binding proteins of the RHO family, and a fourth (CEK1) encodes a putative kinase of the ERK family. Since CEK1 confers a phenotype similar to that of the structurally related S. cerevisiae gene KSS1 but cannot complement a KSS1 defect, it is evident that dominant negative selection can identify proteins that complementation screens would miss. Because dominant negative mutations exert their influence even in wild-type strain backgrounds, this approach should be a general method for the analysis of complex cellular processes in organisms not amenable to direct genetic analysis.
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PMID:Dominant negative selection of heterologous genes: isolation of Candida albicans genes that interfere with Saccharomyces cerevisiae mating factor-induced cell cycle arrest. 140 49

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.
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PMID:Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes. 199 75

Cholecystokinin (CCK) binding to its receptors on guinea pig gastric chief cell membranes was characterized with 125I-COOH terminal octapeptide of CCK (125I-CCK8). Specific binding of 125I-CCK8 to chief cell membranes was maximal at pH 6.0 and 30 degrees C after 180 min of incubation and reversible upon the addition of 10(-7) M unlabeled CCK8. CCK analogs such as CCK8, gastrin-I, and COOH-terminal tetrapeptide of CCK (CCK4) competitively inhibited the labeled CCK8 binding with the half maximal inhibitory concentration of 10(-10) M, 3 X 10(-7) M and 10(-6) M, respectively. Furthermore, guanine nucleotide analogs such as GTP gamma S and Gpp(NH)p also inhibited the labeled CCK8 binding to chief cell membranes. Scatchard analysis of the binding data at pH 6.0 revealed two orders of the binding sites and GTP gamma S decreased the binding by converting two binding sites of the receptors to only one site of lower affinity. These results suggest that there are specific receptors for CCK, which are coupled to a guanine nucleotide regulatory protein on guiea pig gastric chief cell membranes.
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PMID:[Cholecystokinin receptors on guinea pig gastric chief cell membranes]. 212 47

Hormones, neurotransmitter and autacoid receptors, localized on the plasma membrane, do not interact directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger), but control their target systems via activation of an intermediary guanine nucleotide binding protein on G protein, which serves as signal transducer. Traffic of these pathways is regulated via a GTP (on)-GDP (off) switch, which is triggered by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. Receptor desensitization is a main criterion of G protein-coupled receptors with important pharmacological implications. Multiple mechanisms are responsible for the loss of sensitivity that follows against exposure. The process is initiated by uncoupling the receptor from its G protein, which is due to receptor phosphorylation by specific kinases. In the case of the beta-adrenergic receptor, two particular kinases - beta-adrenergic receptor kinase (beta ARK) and protein kinase A--are involved. Further steps of desensitization are receptor sequestration or internalization, an event as rapid and transient as receptor uncoupling, and receptor downregulation, which requires more prolonged agonist exposure. Finally, antagonists are able to induce a receptor-G protein interaction in a reverse manner to agonists. Whereas agonists stimulate both, the GDP dissociation from the G protein and the association of GTP, antagonists markedly decrease GTP association. Moreover, in the turkey erythrocyte adenylyl cyclase system antagonists decrease the GTP-stimulated adenylyl cyclase activity almost at basal levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Role of G protein-mediated signal transduction in molecular pharmacodynamics]. 217 69

The discovery of two distinct succinate thiokinases in mammalian tissues, one (G-STK) specific for GDP/GTP and the other (A-STK) for ADP/ATP, poses the question of their differential metabolic roles. Evidence has suggested that the A-STK functions in the citric acid cycle in the direction of succinyl-CoA breakdown (and ATP formation) whereas one role of the G-STK appears to be the re-cycling of succinate to succinyl-CoA (at the expense of GTP) for the purpose of ketone body activation. A third metabolic participation of succinyl-CoA is in haem biosynthesis. This communication shows that in chemically induced hepatic porphyria, when the demand for succinyl-CoA is increased, it is the level of G-STK only which is elevated, that of A-STK being unaffected. The results implicate G-STK in the provision of succinyl-CoA for haem biosynthesis, a conclusion which is further supported by the observation of a high G-STK/A-STK ratio in bone marrow.
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PMID:Physiological roles of animal succinate thiokinases. Specific association of the guanine nucleotide-linked enzyme with haem biosynthesis. 335 Jan 52

Macrophage-stimulating protein (MSP) is a chemotactic factor that activates the receptor tyrosine kinase RON. The involvement of Ras in MSP-induced signal transduction was investigated. Here we demonstrate that, in RON-transfected MDCK cells, an active GTP-bound form of Ras was rapidly accumulated by MSP treatment and the Ras-guanine nucleotide exchange activity in SOS immunoprecipitates was concomitantly increased. GAP activity was not changed under the same conditions used. Furthermore, the SH2 domain of adaptor protein GRB2, but not Shc, associated with the activated RON-beta chain, and GRB2-SOS complexes translocated from the cytosol to the membrane upon MSP treatment. These results strongly suggest that MSP activates Ras through RON, and that MSP-induced activation of Ras might be controlled by both the enhancement of catalytic exchange activity of SOS and its translocation to the membrane where its target Ras is localized.
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PMID:Macrophage-stimulating protein activates Ras by both activation and translocation of SOS nucleotide exchange factor. 748 76

The importance of PLC activation in cell proliferation is evident from the fact that the hydrolysis of PtdIns(4,5)P2 is one of the early events that follow the interaction of many growth factors and mitogens with their respective receptors. However, the importance of PLC activation is not restricted to proliferation; it is one of the most common transmembrane signaling events elicited by receptors that regulate many other cellular processes, including differentiation, metabolism, secretion, contraction, and sensory perception. It is also clear that cell proliferation signaling does not always require PLC, as indicated by the fact that growth factors such as insulin and CSF-1 do not appear to elicit the hydrolysis of PtdIns(4,5)P2, even though the intracellular domains of their receptors carry a PTK domain and the receptors show topologies very similar to those of the PLC-activating growth factors PDGF, EGF, and FGF. The growth factor-dependent activation of PLC is initiated by the formation of a complex between the receptor PTK and PLC-gamma; the formation of this complex is mediated by a specific interaction between a tyrosine phosphate residue on the intracellular domain of PTK and the SH2 domain of PLC-gamma. The receptor PTK subsequently phosphorylates PLC-gamma, of which two distinct isozymes, PLC-gamma 1 and PLC-gamma 2, have been identified. Proliferation of T cells and B cells in response to the aggregation of their respective cell surface receptors is also accompanied by the activation of PLC-gamma isozymes at an early stage. Unlike growth factor receptors, the T cell and B cell receptors lack intrinsic PTK activity but associate with several non-receptor PTKs of the Src and Syk families. Although the specific kinases are not known, one or more of these enzymes phosphorylate and activate PLC-gamma 1 and PLC-gamma 2. Transduction of growth signals by G protein-coupled receptors such as those for thrombin or bombesin also requires PtdIns(4,5)P2 hydrolysis, which, in this instance, is mediated by PLC-beta isozymes. The PLC-beta subfamily consists of four distinct members: PLC-beta 1, PLC-beta 2, PLC-beta 3, and PLC-beta 4. Agonist interaction with specific G protein-coupled receptors causes the dissociation of Gq proteins into G alpha and G beta gamma subunits and the exchange of GDP bound to G alpha for GTP. The resulting GTP-bound G alpha subunit then activates PLC-beta isoforms by binding to the carboxyl-terminal region of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphoinositide-specific phospholipase C and mitogenic signaling. 749 69

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.
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PMID:Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages. 779 56

Epidermal growth factor (EGF) stimulates adenylyl cyclase in the heart via activation of the stimulatory GTP-binding protein Gs. Therefore, employing peptides corresponding to regions in the cytosolic domain of the EGF receptor, we have investigated the ability of sequences within the EGF receptor to activate Gs. A 13-aa peptide (EGFR-13) corresponding to the juxtamembrane region in the cytosolic domain of the EGF receptor stimulated GTP binding and GTPase activity of Gs. This peptide did not stimulate GTP binding to Gi but increased the GTPase activity of this protein. Additionally, phosphorylation of the protein kinase C site (threonine residue) within EGFR-13 decreased the ability of the peptide to stimulate Gs and increase GTPase activity of Gi. Further, in functional assays of Gs employing S49 cyc- cell membranes, EGFR-13 increased the ability of Gs to stimulate adenylyl cyclase; phospho-EGFR-13 and a 14-aa peptide corresponding to a sequence in the cytosolic domain of the EGF receptor did not alter the functional activity of Gs. Hence, the juxtamembrane region of the EGF receptor can activate Gs and, by stimulating GTPase activity of Gi, inactivates this latter G protein. Phosphorylation of the threonine residue within this region attenuates the activity of the peptide as a modulator of G-protein function.
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PMID:A region in the cytosolic domain of the epidermal growth factor receptor antithetically regulates the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins of adenylyl cyclase. 789 52

Stimulation of platelets by thrombin leads to an increased association of activated phosphoinositide 3-kinase (PI 3-K) with a membrane cytoskeletal fraction (CSK). Activation of PI 3-K is dependent upon GTP-binding protein(s), since PI 3-K in permeabilized platelets is stimulated by GTP gamma S (guanosine 5'-3-O-(thio)triphosphate), and stimulation of platelet cytosolic PI 3-K by GTP gamma S requires a functional small G-protein, Rho. Recent reports indicate that cytosolic PI 3-Ks can also be activated by the beta gamma subunits of heterotrimeric G-proteins (G beta gamma). We now report that the activated PI 3-K that is associated with CSK can be inhibited by a recombinant protein containing the G beta gamma-binding pleckstrin homology domain of beta-adrenergic receptor kinase 1 (beta ARK-PH). Inhibition is blocked by G beta gamma. PI 3-K in nonactivated platelet CSK is activated by GTP gamma S but unaffected by beta ARK-PH or G beta gamma. Western blots indicate that activated platelet CSK contains a novel 110-kDa PI 3-K(gamma) that has been shown to be stimulated by G beta gamma and to lack binding sites for the 85-kDa subunit of conventional PI 3-K. PI 3-K in immunoprecipitates obtained via p85 subunit-directed antibodies can be activated by GTP gamma S but not by G beta gamma. PI 3-K that is stimulatable by G beta gamma remains soluble, as does PI 3-K(gamma), and is unaffected by Rho. In contrast, ADP-ribosylation of Rho present in p85 immunoprecipitates is inhibitory. Further, activation of PI 3-K in permeabilized platelets exposed to thrombin or GTP gamma S is inhibited by beta ARK-PH and/or Rho-specific ADP-ribosylating enzymes. We conclude that Rho and G beta gamma each, respectively, contributes to the activation of different PI 3-Ks (p85-containing heterodimer and PI 3-K (gamma)) in thrombin-stimulated platelets.
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PMID:Sequestration of a G-protein beta gamma subunit or ADP-ribosylation of Rho can inhibit thrombin-induced activation of platelet phosphoinositide 3-kinases. 789 97


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