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F McCormick

Publications and source records attributed to F McCormick.

At least 19 recordsLinked to original sources
PubMed1992

Interaction between the p21ras GTPase activating protein and the insulin receptor.

We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine residues. However, after insulin treatment in the presence of the phosphotyrosine phosphatase inhibitor phenylarsine oxide (PAO), 5-10% of GAP was found to be associated with the insulin receptor, and, in addition, a fraction of total GAP was phosphorylated on tyrosine. Using in vitro binding we showed that the N-terminal part of GAP containing the src-homology domains 2 and 3 (SH2-SH3-SH2 region) is involved in binding to the autophosphorylated insulin receptor beta-chain. In vitro binding between GAP and the autophosphorylated insulin receptor occurred independently of PAO pretreatment. These results suggest that GAP can transiently interact with the insulin receptor after insulin treatment, and this interaction is arrested after PAO pretreatment.

3T3 Cells↗
PubMed1992

Phosphorylation of rap1GAP in vivo and by cAMP-dependent kinase and the cell cycle p34cdc2 kinase in vitro.

rap1GAP is a GTPase activating protein that specifically stimulates the GTP hydrolytic rate of the ras-related protein p21rap1.rap1GAP undergoes post-translational modification that causes a substantial change in its mobility on sodium dodecyl sulfate-polyacrylamide gels. At least part of this modification is due to the phosphorylation. Expression of a rap1GAP cDNA in insect cells labeled with 32Pi resulted in high level incorporation of radioactivity into serine residues of the expressed protein. Purified rap1GAP was phosphorylated in vitro by cAMP-dependent kinase and the cell cycle p34cdc2 kinase. The molar ratio of incorporated phosphate/rap1GAP was approximately 3 by cAMP-dependent kinase and 2 by p34cdc2. The sites of phosphorylation by both kinases were localized to a 100-residue segment contained in the carboxyl-terminal region of the predicted primary structure of rap1GAP. Highly favorable recognition sequences for the two kinases are contained within this fragment and are proposed as the sites of phosphorylation. Treatment of SK-MEL-3 cells with dibutyryl cAMP promoted phosphorylation of rap1GAP in vivo. Based on the results of comparative phosphopeptide mapping the sites of phosphorylation in vivo and in vitro are identical.

Amino Acid Sequence↗
PubMed1992

Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.

3T3 Cells↗
PubMed1992

Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62.

p62 is a tyrosine phosphoprotein that associates with p21ras GTPase-activating protein (GAP). Purification and cDNA cloning of p62 reveal extensive sequence similarity to a putative hnRNP protein, GRP33. Recombinant human p62 purified from insect Sf9 cells binds to DNA and to mRNA and, like many proteins involved in mRNA processing, recombinant p62 is modified by dimethylation on multiple arginine residues. p62 also binds tightly to p21ras GAP in vitro: this binding depends on phosphorylation of p62 on tyrosine residues and occurs through SH2 regions of GAP. These data suggest that p120-GAP and p62 play a role in some aspect of mRNA processing or utilization and that this role may be regulated by tyrosine phosphorylation, and indirectly, by p21ras.

3T3 Cells↗
PubMed1992

Coupling of ras p21 signalling and GTP hydrolysis by GTPase activating proteins.

Ras p21 proteins cycle between inactive, GDP-bound forms and active GTP-bound forms. Hydrolysis of bound GTP to GDP is mediated by proteins referred to as GAPs, two forms of which have been described. The first, p120-GAP, contains regions of homologies with tyrosine kinase oncogenes, and interacts with tyrosine phosphoproteins as well as with ras proteins; p120-GAP may therefore connect signalling pathways that involve tyrosine kinase and ras p21 proteins. The second type of GAP is the product of the neurofibromatosis type 1 gene (NF1-GAP). This is a protein of 325,000 Da that is defective in patients with NF1; NF1-GAP is regulated by signalling lipids, and may serve to connect ras p21 with phospholipid second messenger systems. The significance of ras p21 interaction with distinct GAPs is discussed.

Animals↗
PubMed1992

p21rasGAP association with Fyn, Lyn, and Yes in thrombin-activated platelets.

Activation of platelets by thrombin and other physiological agonists leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins (Ferrell, J. E., and Martin, G. S. (1988) Mol. Cell. Biol. 8, 3606-3610; Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 901-905; Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091). To date, none of the tyrosine kinases that are involved in platelet activation, nor the substrates that are phosphorylated in response to agonists, have been identified. A "kinase trapping" strategy, designed to take advantage of the stability of known tyrosine kinase-substrate interactions, was employed to address both issues. p21rasGAP antibodies were used to examine the phosphorylated state of GAP in agonist-treated platelets and to isolate potential GAP-kinase complexes. We show that GAP and two proteins of 59 and 68 kDa are phosphorylated on tyrosine after thrombin stimulation and that three Src-related protein tyrosine kinases, Fyn, Lyn and Yes, are associated with GAP in complexes, detectable only after agonist stimulation. The thrombin-dependent detection of these kinases in GAP immunoprecipitates suggests that thrombin may either induce the formation of these complexes or activate kinases that are associated with GAP prior to, or following, agonist stimulation. This approach of "trapping" kinases bound to their substrates will be useful in identifying non-receptor tyrosine kinases involved in signaling pathways. Furthermore, although GAP phosphorylation has been previously implicated in growth factor signaling pathways, this is the first example of its involvement downstream from a G-protein-coupled receptor.

Blood Platelets↗
PubMed1992

GAP domains responsible for ras p21-dependent inhibition of muscarinic atrial K+ channel currents.

The interaction between the low molecular weight G protein ras p21 and a guanosine triphosphatase activating protein (GAP) uncouples a heterotrimeric G protein (Gk) from muscarinic receptors. Through the use of isolated atrial cell membranes and genetically engineered GAP deletion mutants, the src homology regions (SH2-SH3) at the amino terminus of GAP have been identified as the domains responsible for this effect. Deletion of the domain required to stimulate the guanosine triphosphatase activity of ras p21 relieves the requirement for ras p21 in this system. A model is presented that suggests that ras p21 induces a conformational change in GAP, which allows the SH2-SH3 regions of GAP to function.

Animals↗
PubMed1992

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

Amino Acid Sequence↗
PubMed1992

Specific changes of Ras GTPase-activating protein (GAP) and a GAP-associated p62 protein during calcium-induced keratinocyte differentiation.

Induction of tyrosine phosphorylation occurs as an early and specific event in keratinocyte differentiation. A set of tyrosine-phosphorylated substrates which transduce mitogenic signals by tyrosine kinases has previously been identified. We show here that of these substrates, the Ras GTPase-activating protein, GAP, is specifically affected during calcium-induced keratinocyte differentiation. As early as 10 min after calcium addition to cultured primary mouse keratinocytes, GAP associates with tyrosine-phosphorylated proteins and translocates to the membrane. In addition, a GAP-associated protein of approximately 62 kDa (p62) becomes rapidly and heavily tyrosine phosphorylated in both membrane and cytosolic fractions. This protein corresponds to the major tyrosine-phosphorylated protein that is induced in differentiating keratinocytes as early as 5 min after calcium addition. p62 phosphorylation was not observed after exposure of these cells to epidermal growth factor, phorbol ester, or transforming growth factor beta. In contrast, PLC gamma and P13K were tyrosine phosphorylated after epidermal growth factor, but not calcium, stimulation. Thus, changes of Ras GAP and an associated p62 protein occur as early and specific events in keratinocyte differentiation and appear to involve a calcium-induced tyrosine kinase.

3T3 Cells↗
PubMed1992

GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion.

The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.

3T3 Cells↗
PubMed1992

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

Amino Acid Sequence↗
PubMed1992

Interactions between p21ras proteins and their GTPase activating proteins.

Two proteins that regulate p21ras GTPase activity have been identified. These proteins interact with a region of ras p21 that is necessary for p21ras function and may themselves be components of signalling complexes. The first of these proteins to be identified, GAP, contains domains that interact with receptor tyrosine kinases and other tyrosine phosphoproteins, providing a direct link between signalling pathways involving these proteins and p21ras. The second, the product of the NF1 gene, is less well characterized but seems to connect p21ras to other signalling pathways which are perturbed in the NF1 disease. The ability of p21ras to interact with GAP may be compromised by competitive binding to the product of the Ki-rev1 gene, p21rap1. This competition for binding to GAP, or other proteins that interact with the effector site of ras p21, may explain the ability of Ki-rev1 to suppress cellular transformation by ras oncogenes.

GTPase-Activating Proteins↗
PubMed1992

GTPase activating proteins.

Since Ras proteins negotiate many signalling pathways leading to cell growth or differentiation, the regulation of Ras activity is vital to cellular health. Ras activity, which derives from a collaboration between Ras and GTP, is terminated by the GTPase activating protein (GAP)-catalyzed hydrolysis of the GTP. Hence, a simple regulatory scheme emerges: extracellular signals control Ras activity via membrane receptors and GAPs. However, the signalling scenario is probably not so simple. In looking for factors which interpret Ras activity, researchers have been led to the same factors which also regulate Ras activity, namely the GAPs. Therefore, it may be that Ras proteins are actually regulators of GAPs.

Animals↗
PubMed1992

Prevalence of N-ras mutations in children with myelodysplastic syndromes and acute myeloid leukemia.

The ras proto-oncogene family encodes a group of 21 kDa nucleotide-binding proteins. Activating mutations of ras genes are associated with certain types of malignancies, indicating that they are related in some way to the malignant process. We have examined bone marrow cells from nine children with myelodysplastic syndromes (MDS) and 35 with acute myeloid leukemia (AML) for activating point mutations of ras genes by in vitro amplification using polymerase chain reaction (PCR), oligonucleotide hybridization and sequencing of PCR products. We found N-ras mutations in cells from 3 of 9 children (33%) with MDS and only 2 of 35 children with AML (6%; 95% confidence interval is 0.7-19%). All mutations the second nucleotide of codon 12 or the first nucleotide of codon 61 of N-ras. There was no apparent correlation with clinical or laboratory characteristics, including karyotype; however, an association of N-ras activation with the most aggressive type of MDS was noted. Among the patients with MDS, 2 of 6 with monosomy 7 had N-ras mutations; however, three children with monosomy 7 which presented with AML lacked ras mutations. One patient was studied at time of diagnosis of MDS and again after progression to AML. At the preleukemic stage of disease, an N-ras mutation was identified; however, after development of AML this mutation was not present in the leukemic clone. In conclusion, these data show that ras mutations, while not necessary for leukemic transformation, may be important for the initiation of preleukemias evolving into overt AML.

Adolescent↗
PubMed1992

Association of a tyrosine kinase activity with GAP complexes in v-src transformed fibroblasts.

p21ras GAP is phosphorylated on tyrosine residues and associates with 62 kDa and 190 kDa tyrosine phosphorylated proteins in v-src-transformed fibroblasts. We were interested in identifying the tyrosine kinase responsible for phosphorylation of GAP and the two associated proteins. Here, we report that GAP-immunoprecipitates from v-src transformed cells contain a tyrosine kinase activity that phosphorylates GAP, p62 and p190. Tryptic peptide analysis indicated that the sites phosphorylated in vitro and in vivo are indistinguishable, suggesting that the precipitated kinase could be responsible for tyrosine phosphorylation of GAP in vivo. The GAP-associated kinase activity might be due to v-src itself, because pp60v-src is able to associate with GAP in vitro and GAP can be phosphorylated by pp60v-src immunecomplexes.

3T3 Cells↗
PubMed1991

Heterogeneous amino acids in Ras and Rap1A specifying sensitivity to GAP proteins.

Guanosine triphosphatase (GTPase) activity of Ras is increased by interaction with Ras-GAP (GTPase-activating protein) or with the GAP-related domain of the type 1 neurofibromatosis protein (NF1-GRD), but Ras is not affected by interaction with cytoplasmic and membrane forms of Rap-GAP; Rap1A, whose effector function can suppress transformation by Ras, is sensitive to both forms of Rap-GAP and resistant to Ras-GAP and NF1-GRD. A series of chimeric proteins composed of portions of Ras and Rap were constructed; some were sensitive to Ras-GAP but resistant to NF1-GRD, and others were sensitive to cytoplasmic Rap-GAP but resistant to membrane Rap-GAP. Sensitivity of chimeras to Ras-GAP and cytoplasmic Rap-GAP was mediated by amino acids that are carboxyl-terminal to the effector region. Residues 61 to 65 of Ras conferred Ras-GAP sensitivity, but a larger number of Rap1A residues were required for sensitivity to cytoplasmic Rap-GAP. Chimeras carrying the Ras effector region that were sensitive only to Ras-GAP or only to cytoplasmic Rap-GAP transformed NIH 3T3 cells poorly. Thus, distinct amino acids of Ras and Rap1A mediate sensitivity to each of the proteins with GAP activity, and transforming potential of Ras and sensitivity of Ras to Ras-GAP are at least partially independent properties.

Cell Membrane↗