title: ARAP3 is transiently tyrosine phosphorylated in cells attaching to fibronectin and inhibits cell spreading in a RhoGAP-dependent manner.
abstract: ARAP3 is a GTPase activating protein (GAP) for Rho and Arf GTPases that is implicated in phosphoinositide 3-kinase (PI 3-kinase) signalling pathways controlling lamellipodia formation and actin stress fibre assembly. We have identified ARAP3 as a phosphorylated target of protein tyrosine kinases. In cells, ARAP3 was tyrosine phosphorylated when co-expressed with Src-family kinases (SFKs), upon stimulation with growth factors and during adhesion to the extracellular matrix (ECM) substrate fibronectin. Adhesion-induced phosphorylation of ARAP3 was suppressed by selective inhibitors of Src-family kinases and PI 3-kinase and by a Src dominant interfering mutant. Inducible expression of ARAP3 in HEK293 epithelial cells resulted in increased cell rounding, membrane process formation and cell clustering on ECM substrates. In contrast, ARAP3 dramatically slowed the kinetics of cell spreading on fibronectin but had no effect on cell adhesion. These effects of ARAP3 required a functional Rho GAP domain and were associated with reduced cellular levels of active RhoA and Rac1 but did not require the sterile alpha motif (SAM) or Arf GAP domains. Mutation of two phosphorylation sites, Y1399 and Y1404, enhanced some ARAP3 activities, suggesting that ARAP3 may be negatively regulated by phosphorylation on these tyrosine residues. These results implicate ARAP3 in integrin-mediated tyrosine kinase signalling pathways controlling Rho GTPases and cell spreading.
authors.affiliation: Department of Surgery, University of Melbourne, Level 5 Clinical Sciences Building, Royal Melbourne Hospital, VIC 3050, Australia.
authors.forename: Stacey T T
authors.initials: ST
authors.lastname: I
authors.forename: Zhongzhen
authors.initials: Z
authors.lastname: Nie
authors.forename: Ashley
authors.initials: A
authors.lastname: Stewart
authors.forename: Meri
authors.initials: M
authors.lastname: Najdovska
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Hall
authors.forename: Hong
authors.initials: H
authors.lastname: He
authors.forename: Paul A
authors.initials: PA
authors.lastname: Randazzo
authors.forename: Peter
authors.initials: P
authors.lastname: Lock
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007700:Kinetics; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: C446512:ARAP3 protein, human; D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; D005353:Fibronectins; D020690:GTPase-Activating Proteins; D011994:Recombinant Proteins; C085379:rho GTPase-activating protein; D014443:Tyrosine; D004247:DNA; D019061:src-Family Kinases; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein