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.

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

“…Neither ArfGAP activity nor the FAK-binding SHD1 domain was essential for PKL phosphorylation ( Figure 5). The precise role for PKL Arf-GAP activity in regulating cell protrusiveness, spreading and motility is unclear, although it is intriguing that FAK-and Src-family kinases interact with and phosphorylate a number of Arf-GAP proteins including ASAP1/2, ARAP3 and GIT1/GIT2 (Brown et al, 1998b;Andreev et al, 1999;Bagrodia et al, 1999;Zhao et al, 2000b;Haendeler et al, 2003;Randazzo and Hirsch, 2004;Stacey et al, 2004;Van Nieuw Amerongen et al, 2004;Yin et al, 2004). Additional work will be required to determine the precise role for PKL phosphorylation in generating a focal adhesion competent molecule.…”

“…The p21 GTPases themselves are regulated by guanine nucleotide exchange factors (GEFs) that turn them on, GTPase activating proteins (GAPs) that turn them off, and guanine nucleotide dissociation inhibitors that can function as sequestering agents (Suetsugu and Takenawa, 2003;Burridge and Wennerberg, 2004;Raftopoulou and Hall, 2004). The activities of many of these proteins have been found to be regulated by FAK and Src-family kinases (Hildebrand et al, 1996;Han et al, 1997;Kiyono et al, 2000;Haskell et al, 2001;Tu et al, 2003;Zhai et al, 2003;Stacey et al, 2004).…”

“…The p21 GTPases themselves are regulated by guanine nucleotide exchange factors (GEFs) that turn them on, GTPase activating proteins (GAPs) that turn them off, and guanine nucleotide dissociation inhibitors that can function as sequestering agents (Suetsugu and Takenawa, 2003;Burridge and Wennerberg, 2004;Raftopoulou and Hall, 2004). The activities of many of these proteins have been found to be regulated by FAK and Src-family kinases (Hildebrand et al, 1996;Han et al, 1997;Kiyono et al, 2000;Haskell et al, 2001;Tu et al, 2003;Zhai et al, 2003;Stacey et al, 2004).The pathways of activation and signaling from the p21-activated protein kinase (PAK) typify the sophisticated mechanisms developed by cells to respond to extracellular cues (Bokoch, 2003). In response to cell attachment and/or growth factor stimulation, PAK is activated and signals to Galisteo et al, 1996;Lu et al, 1997;Sells et al, 1997;Kiosses et al, 2002), the Cdc42/Rac GEF PIX (Bagrodia et al, 1998;Zhao et al, 2000a), the paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT) family of ArfGAPs Premont et al, 2000;Brown et al, 2002;Manabe Ri et al, 2002), and the molecular scaffold protein paxillin Zhao et al, 2000b;Hashimoto et al, 2001;West et al, 2001;Brown et al, 2002).…”

The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner

“…It was also proposed before that SHIP2 is involved in the regulation of the actin cytoskeleton and cell adhesion, like Arap3, and that it interacts with multiple proteins in the cytoskeleton network [5,6,24,25,28]. It will therefore be interesting to see which of these proteins are found in the same complex together, and what exactly is the role of all these different interactions in the complex signaling pathways that eventually lead to cell adhesion.…”

“…Upon binding of PI(3,4,5,)P 3 lipids to one of its pleckstrin homology (PH) domains, Arap3 translocates to the plasma membrane and is activated to serve as a dual GTPase activating protein (GAP) for Arf and Rho G-proteins [4]. Arap3 is implicated in the regulation of the actin cytoskeleton, lamellipodia formation and cell spreading [5,6]. In addition, Arap3 contains an RA domain that binds specifically to the small G-protein Rap1 and a SAM domain of unknown function [4] (Fig.…”