CELLBIOLOGY Tyrosine Phosphorylation of the Ga-Interacting Protein GIV Promotes Activation of Phosphoinositide 3-Kinase During Cell Migration Changsheng Lin, 1 Jason Ear, 1 Yelena Pavlova, 1 Yash Mittal, 1 Irina Kufareva, 2 Majid Ghassemian, 3 Ruben Abagyan, 2 Mikel Garcia-Marcos, 4 Pradipta Ghosh 1 * GIV (Ga-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation downstream of multiple growth factor– and G protein (heterotrimeric guanosine 5′-triphosphate–binding protein)–coupled receptors to trigger cell migration and cancer invasion. We demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at tyrosine-1764 and tyrosine-1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the amino- and carboxyl-terminal Src homology 2 domains of p85a, a regulatory subunit of PI3K; stabilized receptor association with PI3K; and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85a increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIV-PI3K interaction a potential therapeutic target within the PI3K-Akt pathway. INTRODUCTION GIV (Ga-interacting vesicle-associated protein; also known as Girdin) is a multidomain protein that is required for growth factors [epidermal growth factor (EGF) (1, 2), insulin-like growth factor (IGF) (3), vascular endothe- lial growth factor (VEGF) (4), and insulin (5–8)] to enhance Akt activation in a phosphoinositide 3-kinase (PI3K)–dependent manner (5), remodel actin, and trigger cell migration. GIV also enhances Akt activation down- stream of heterotrimeric guanosine 5′-triphosphate–binding protein (G protein)–coupled receptors (GPCRs) (7–9). Working downstream of growth factor receptor tyrosine kinases and GPCRs, GIV enhances Akt signals during diverse biological processes, including epithelial wound healing, macrophage chemotaxis, development, autophagy, tumor angiogenesis, tumor cell migration, and cancer invasion and metastasis (1–4, 6–9). We previously demonstrated that GIV is a non-receptor guanine nu- cleotide exchange factor (GEF) for Ga i (7) and that GIV directly binds ligand-activated EGF receptor (EGFR) (2). By linking G protein signal- ing to EGFR and assembling a Ga i -GIV-EGFR signaling complex, GIV enhances EGFR autophosphorylation, prolongs receptor association with the plasma membrane, and enhances Akt signals from the plasma mem- brane to trigger cell migration. However, the underlying mechanism of how multiple receptors use GIV for Akt enhancement has remained unknown. Because tyrosine phosphorylation–based signaling pathways are major activators of the PI3K-Akt pathway and because GIV responds to multiple growth factor receptor tyrosine kinases to enhance Akt activation in a PI3K-dependent manner (5), we investigated whether GIV is a substrate for tyrosine kinases and whether such phosphorylation would regulate its ability to activate PI3K. RESULTS GIV is phosphorylated by receptor and non-receptor tyrosine kinases To investigate whether GIV is phosphorylated by tyrosine kinases, we performed in vitro kinase assays using recombinant growth factor re- ceptor tyrosine kinases [EGFR, PDGFR (platelet-derived growth fac- tor receptor), and VEGFR (VEGF receptor)] and the C-terminal domain of GIV (His–GIV-CT; amino acids 1660 to 1870) and determined the extent of GIV phosphorylation by immunoblotting for phosphotyrosine (pTyr). We examined the C terminus of GIV because GIV directly binds EGFR through this domain (2). All three receptor tyrosine kinases phos- phorylated GIV to a similar extent (Fig. 1A), as did TrkA (tyrosine kinase A receptor), the receptor for nerve growth factor (NGF) (fig. S1A). Simi- lar results were obtained when the C-terminal domain of His-GIV was subjected to kinase assays with recombinant c-Src, a non-receptor tyrosine kinase (Fig. 1A). To determine whether GIV was tyrosine phosphorylated in cells, we immunoprecipitated endogenous GIV from EGF-treated HeLa cells and immunoblotted for pTyr and GIV and found that GIV was phos- phorylated on tyrosine residues in cells treated with EGF (Fig. 1B, lanes 1 and 2). When tyrosine phosphoproteins were immunoprecipitated from HeLa cells with a pTyr antibody, GIV was detected in the immunopre- cipitates (Fig. 1B, lanes 3 and 4), thus confirming that GIV is a tyrosine phosphoprotein in cells treated with EGF. Similar results were also ob- served in HeLa cells after insulin stimulation (fig. S1B). We conclude that GIV is a tyrosine phosphoprotein that is a common target of receptor and non-receptor tyrosine kinases. We performed mass spectrometry (MS) on in vitro EGFR-phosphorylated C-terminal domain of GIV and identified Tyr 1764 and Tyr 1798 as the sites of phosphorylation (Fig. 1C and fig. S2). A phylogenetic analysis of GIV 1 Department of Medicine, University of California, San Diego, School of Med- icine, La Jolla, CA 92093, USA. 2 School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA. 3 De- partment of Chemistry and Biochemistry, University of California, San Diego, School of Medicine, La Jolla, CA 92093, USA. 4 Department of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92093, USA. *To whom correspondence should be addressed. E-mail: prghosh@ucsd.edu RESEARCHARTICLE www.SCIENCESIGNALING.org 27 September 2011 Vol 4 Issue 192 ra64 1 on December 18, 2014 http://stke.sciencemag.org/ Downloaded from