A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

Marshall, Mark S.; Hill, W. S.; Ng, Assunta S.; Vogel, U. S.; Schaber, Michael D.; Scolnick, Edward M.; Dixon, Richard A. F.; Sigal, Irving S.; Gibbs, Jackson B.

doi:10.1002/j.1460-2075.1989.tb03480.x

Cited by 159 publications

(62 citation statements)

References 25 publications

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“…The wild-type GAP cDNA contains the complete coding sequence of human type 1 GAP. It contains a hydrophobic NH2 terminus of about 180 amino acids, the src-homology regions (SH2-SH3) directly adjacent to it, and the catalytic domain responsible for activation of the p2lras GTPase activity in the COOHterminal part of the protein (20). GAP32 lacks a substantial part of the region for p2lras interaction.…”

Section: Resultsmentioning

confidence: 99%

“…The COOHterminal domain of GAP is responsible for this catalytic effect on p2lr's GTPase activity (20). In addition, through its src-homology domains (SH2-SH3), GAP can associate with tyrosine-phosphorylated proteins (2,25) such as the plateletderived growth factor receptor (18), epidermal growth factor receptor (19), the insulin receptor (28), v-src (7,29), and two proteins of 190 and 62 kDa (12).…”

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confidence: 99%

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

Medema¹,

Laat²,

Martin³

et al. 1992

Mol. Cell. Biol.

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The p2l' GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p2l1. 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 p2l' and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction offos promoter activity similar to induction by activated p21'. Expression of a full-length GAP construct had no effect on the activity of thefos promoter. Activation of thefos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21', Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21' activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p2l1, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p2l'. 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 p21' but cooperate with another signal coming from active p2l'. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21' but that additional signals coming from p21' are required for them to function.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Medema¹,

Laat²,

Martin³

et al. 1992

Mol. Cell. Biol.

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“…SH2 domains are not required for catalytic activity of enzymes in which they are found (12)(13)(14)(15)(16), but mutations in this domain can have profound effects on biological activity (6,(17)(18)(19)(20). PLC-y, GAP, and the src family of tyrosine kinases have recently been shown to bind to growth-factor receptors after ligand binding (21)(22)(23)(24)(25)(26), and the crk protein binds phosphotyrosine-containing proteins and protein kinase activities (27,28), raising the possibility that SH2 domains might mediate protein-protein interactions important to growth control.…”

mentioning

confidence: 99%

The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity.

Mayer

Jackson

Baltimore

1991

Proc. Natl. Acad. Sci. U.S.A.

207

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Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2). We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins. abI SH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution. The SH2 probe bound almost exclusively to tyrosinephosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins. Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction. These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependant binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.The abl gene product is a member of the nonreceptor class of protein-tyrosine kinases and can induce malignant transformation when altered during retroviral transduction or chromosomal translocation (1-4). The amino terminus of the abl kinase contains an 80-amino acid domain, termed src homology region 2 (SH2) (5, 6) that is found in all known nonreceptor tyrosine kinases, a phosphatidylinositol-specific phospholipase C (PLC-'y), the ras GTPase activator protein (GAP), and the crk oncogene product (7-12); each of these proteins has been implicated in growth-control pathways. SH2 domains are not required for catalytic activity of enzymes in which they are found (12-16), but mutations in this domain can have profound effects on biological activity (6,(17)(18)(19)(20). PLC-y, GAP, and the src family of tyrosine kinases have recently been shown to bind to growth-factor receptors after ligand binding (21)(22)(23)(24)(25)(26), and the crk protein binds phosphotyrosine-containing proteins and protein kinase activities (27, 28), raising the possibility that SH2 domains might mediate protein-protein interactions important to growth control. We therefore investigated whether the isolated SH2 domain of abi could specifically bind to cellular proteins in vitro. MATERIALS AND METHODSGeneration of Bacterially Expressed SH2 Probe. A BamHI 12-mer linker was inserted at the HincII site at amino acid 144 of type IV c-abl, and the 0.3-kilobase (kb) BamHI-HinPI fragment was subcloned into pGEX-2T (29) cut with BamHI and Sma I. Bacteria were grown and proteins purified essentially as described (29).Bacterial proteins were coupled at room temperature for 4 hr with biotinamidocaproate N-hydroxysuccinimide ester (Sigma) in 100 mM sodium borate, pH 8.8, at a bacterial peptide concentration of 3 mg/ml and biotin ester at 50 Ag/mg of peptide. Biotinylated peptide was purified by gel filtration.Binding Assays. Tissue-culture cells were lysed on ice in Triton extraction buffer [10 mM Tris, pH 7.4/150 mM NaCl/5 mM EDTA/10% (vol/vol) glycerol/1% Triton X-100/1 mM phenylmethylsu...

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“…p120 cAP is phosphorylated by receptors and non-receptor tyrosine kinases with Y723 being identified as a site ofphosphorylation [19,20]. This lies within the region which, has been suggested on the basis of deletional analysis as a site of interaction with Ras [15]. The results of this study have implied that the region 700-750 is important for GAP activity as a negative regulator or as an effector protein for Ras.…”

Section: Introductionmentioning

confidence: 63%

“…However, it is possible that GAP may also play the role of effector molecule for Ras, as, for example, the Ras' GTP" GAP ternary complex is essential for oocyte maturation [14]. The interaction site on GAP for Ras has been approximately mapped by deletional studies to a region towards the C-terminus of the molecule [15]. The C-terminal 344 amino acids of GAP have been shown to stimulate the isomerisation step which precedes GTP hydrolysis by Ras 200-fold [16] whereas the full-length protein causes an increase of about 105-fold.…”

Section: Introductionmentioning

confidence: 99%