Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases

Abstract

The critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. As microinjection of antibodies against p21ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21ras to transmit their mitogenic signals. No biochemical link has been established, however, between tyrosine kinases and p21ras. We have identified a non-catalytic domain of cytoplasmic tyrosine kinases, SH2, that regulates the activity and specificity of the kinase domain. The presence of two adjacent SH2 domains in the p21ras GTPase-activating protein (GAP) indicates that GAP might interact directly with tyrosine kinases. Here we show that GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of the v-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. Our results suggest a mechanism by which tyrosine kinases might modify p21ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction.

Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes

“…epithelial cells by Ras. A possible explanation for our results stems from studies showing that Lck and Src phosphorylate Ras-GTPase activating protein (Ras-GAP) (Ellis et al, 1990(Ellis et al, , 1991Park et al, 1992;Amrein et al, 1992). If this phosphorylation is associated with increased activity of Ras-GAP, one would expect to increase the GTPase activity towards Ras and therefore induce a downregulation of Rasmediated signal transduction (van der Geer et al, 1997; Sche zek et al, 1997).…”

Increasing complexity of Ras signaling

“…A current working model proposes that Ras-GTP binding to the COOH-terminal catalytic domain would expose the protein-ligand binding motifs present in the NH 2 -terminus, and the subsequent association of components with the NH 2 -terminus would activate a signaling function. The identi®cation of other p120 GAP-binding proteins in addition to p190 Rho GAP, (e.g., p62 dok , G3BP) provide support for such a scenario (Settleman et al, 1992;Yamanashi and Baltimore, 1997;Ellis et al, 1990;Parker et al, 1996).…”

RAFTK/Pyk2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell invasion

“…Interestingly, p190-B gene expression is regulated during mammary development and p190-B is upregulated in a subset of experimentally-induced rodent tumors (personal communication, Geetika Chakravarty and Je Rosen, Baylor University School of Medicine). This suggests that formation of the RasGAP-p190 complex may be a critical step in the transformation process (Ellis et al, 1990).…”

“…Since it has been shown that growth factor stimulation induces complex formation between p190 and RasGAP (Ellis et al, 1990;Kaplan et al, 1990;Reedijk et al, 1990), we examined the presence of RasGAP in the HRG-stimulated complex of RAFTK. T47D cells were stimulated with HRG for 2 h, and the lysates from the stimulated and unstimulated cells were immunoprecipitated with anti-RAFTK antibodies or with NRS as a control.…”