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 Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two 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 mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived 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 correlated 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 suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)
McGlade
J
J
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Brunkhorst
B
B
Anderson
D
D
Mbamalu
G
G
Settleman
J
J
Dedhar
S
S
Rozakis-Adcock
M
M
Chen
L B
LB
Pawson
T
T
D000199:Actins / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology*; D004268:DNA-Binding Proteins; D005353:Fibronectins / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins
D000199:Actins; C513040:Arhgap35 protein, rat; D004268:DNA-Binding Proteins; D005353:Fibronectins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D012097:Repressor Proteins; D020703:ras GTPase-Activating Proteins; D017479:Receptors, Platelet-Derived Growth Factor; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins