THE N-TERMINAL REGION OF GAP REGULATES CYTOSKELETAL STRUCTURE AND CELL-ADHESION

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ra s, GAP binds stably to autophosphorylated betaPDGF receptors, and to t wo cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that medi ates these interactions with cellular phosphoproteins, and to investig ate the biological significance of these complexes, a truncated GAP po lypeptide (GAP-N) containing residues 1-445 was stably expressed in Ra t-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N w ith PDGF induced association of GAP-N with the betaPDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated betaPDGF rece ptor in vivo. GAP-N bound constitutively to p190 in both serum-deprive d and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlat ed with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results sugge st that the N-terminal domain of GAP can direct interactions with cell ular phosphoproteins in vivo, and thereby exert an effector function w hich modulates the cytoskeleton and cell adhesion. This effect of GAP- N on the cytoskeleton correlates with its association with p190, and m ay therefore result from regulation of Rho/Rac GTPases by the GAP-p190 complex. GAP may therefore couple growth factors to control of cell s hape and attachment.