Research ArticleCell Biology

Tyrosine Phosphorylation of the Gα-Interacting Protein GIV Promotes Activation of Phosphoinositide 3-Kinase During Cell Migration

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Science Signaling  27 Sep 2011:
Vol. 4, Issue 192, pp. ra64
DOI: 10.1126/scisignal.2002049

Abstract

GIV (Gα-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 p85α, 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 p85α 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 (Gα-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 endothelial growth factor (VEGF) (4), and insulin (58)] to enhance Akt activation in a phosphoinositide 3-kinase (PI3K)–dependent manner (5), remodel actin, and trigger cell migration. GIV also enhances Akt activation downstream of heterotrimeric guanosine 5′-triphosphate–binding protein (G protein)–coupled receptors (GPCRs) (79). 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 (14, 69).

We previously demonstrated that GIV is a non-receptor guanine nucleotide exchange factor (GEF) for Gαi (7) and that GIV directly binds ligand-activated EGF receptor (EGFR) (2). By linking G protein signaling to EGFR and assembling a Gαi-GIV-EGFR signaling complex, GIV enhances EGFR autophosphorylation, prolongs receptor association with the plasma membrane, and enhances Akt signals from the plasma membrane 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 receptor tyrosine kinases [EGFR, PDGFR (platelet-derived growth factor 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 phosphorylated GIV to a similar extent (Fig. 1A), as did TrkA (tyrosine kinase A receptor), the receptor for nerve growth factor (NGF) (fig. S1A). Similar 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 phosphorylated 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 immunoprecipitates (Fig. 1B, lanes 3 and 4), thus confirming that GIV is a tyrosine phosphoprotein in cells treated with EGF. Similar results were also observed 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.

Fig. 1

GIV is phosphorylated on Tyr1764 and Tyr1798 by receptor and non-receptor tyrosine kinases. (A) In vitro kinase assays with the indicated recombinant tyrosine kinases were carried out on the His-tagged C-terminal domain of GIV (His–GIV-CT) and immunoblotted (IB) for tyrosine phosphorylation. (B) GIV (lane 2; pre-immune IgG, lane 1) and phosphotyrosine (pTyr) proteins (lane 4; pre-immune IgG, lane 3) were immunoprecipitated from EGF-treated HeLa cells and analyzed by two-color immunoblotting for GIV and pTyr. Single-channel images for GIV and pTyr are displayed in grayscale, and the overlay of GIV (red) and pTyr (green) images is displayed in the merged panels. (C) Tyr1764 and Tyr1798 are located in the C terminus within the EGFR, Akt, and actin-binding domains (orange) and were the only sites of tyrosine phosphorylation identified by phosphoproteomic analysis (fig. S3). (D) In vitro kinase assays with recombinant EGFR (top panel) and c-Src (middle panel) on wild-type (WT), Y1764F, Y1798F, and Y1764,Y1798F mutants of His–GIV-CT (Ponceau S, bottom panel) were analyzed for tyrosine phosphorylation. (E) COS-7 cells expressing FLAG-tagged GIV-WT (GIV-WT–FLAG), a tyrosine phosphorylation–deficient mutant (GIV-YF–FLAG), or vector alone were serum-starved or stimulated with EGF. FLAG immunoprecipitates (top) were analyzed by two-color immunoblotting for GIV (red) and pTyr (green). (F) COS-7 cells expressing c-Src–HA and GIV-WT–FLAG, GIV-YF–FLAG, or vector control were serum-starved, incubated with or without PP2, and stimulated with LPA. Src was inhibited by PP2 as determined by decreased phosphorylation of Src at Tyr416. FLAG immune complexes (top panels) were analyzed by two-color immunoblotting for GIV (red) and pTyr (green). Tyrosine phosphorylation occurred in GIV-WT–FLAG–expressing cells upon ligand stimulation and in the absence of PP2 (lane 3). GIV-WT–FLAG was tyrosine phosphorylated (Merge) after LPA stimulation (lane 3) but not when Src was inhibited with PP2 (lane 5). GIV-YF is not tyrosine phosphorylated (lanes 4 and 6).

We performed mass spectrometry (MS) on in vitro EGFR-phosphorylated C-terminal domain of GIV and identified Tyr1764 and Tyr1798 as the sites of phosphorylation (Fig. 1C and fig. S2). A phylogenetic analysis of GIV revealed that these two tyrosine residues are conserved in birds and mammals, whereas both are absent in fish or lower animals (fig. S2D). These tyrosine residues were previously identified as major phosphosites in cells (http://www.Phosphosite.org) on the basis of the curated information from several independently conducted, high-throughput phosphoproteomic studies (1014) (fig. S2A). In addition, multiple kinase prediction programs identified the sequences flanking these tyrosine residues as suitable substrates for both receptor and non-receptor tyrosine kinases (fig. S2A). Whereas the sequence flanking Tyr1764 was predicted to make that site favorable for EGFR, the sequence flanking Tyr1798 appeared favorable for Src (fig. S2, B and C). In vitro kinase assays using the C-terminal domain of GIV with phenylalanine substitutions for either or both of the tyrosine residues confirmed that both Tyr1764 and Tyr1798 were substrates for EGFR and Src (Fig. 1D). The Tyr1798→Phe (Y1798F) mutant was a better substrate for EGFR, whereas the Tyr1764→Phe (Y1764F) mutant was a better substrate for Src. Because both kinases failed to phosphorylate the mutant lacking both tyrosine residues, we conclude that these residues accounted for the observed in vitro phosphorylation (Fig. 1A). To discern whether these two C-terminal tyrosine residues also account for the phosphorylation of full-length GIV observed in cells in Fig. 1B, we determined the tyrosine phosphorylation status of wild-type GIV (GIV-WT) or a GIV mutant in which both tyrosine residues were replaced by phenylalanines (GIV-YF) in COS-7 cells. Upon EGF stimulation, wild-type GIV, but not the tyrosine phosphorylation–deficient mutant, was tyrosine phosphorylated (Fig. 1E), suggesting that Tyr1764 and Tyr1798 may be the major sites of tyrosine phosphorylation in GIV. Together, these results demonstrate that both EGFR and Src can phosphorylate GIV at two C-terminally located tyrosine residues in vitro and that these residues are phosphorylated after ligand stimulation in cells.

Receptor and non-receptor tyrosine kinases cooperatively phosphorylate GIV after stimulation of receptor tyrosine kinases and GPCRs

Because EGF stimulation results in activation of both EGFR and Src (15), we asked whether ligand-dependent tyrosine phosphorylation of GIV is mediated by both receptor and non-receptor kinases. We used a selective inhibitor for Src family kinases, protein phosphatase 2 (PP2) (16), in cellular phosphorylation assays. Upon EGF stimulation, wild-type GIV was phosphorylated in the presence and absence of PP2 (fig. S3), suggesting that GIV is tyrosine phosphorylated, likely by EGFR, when Src family kinases are inhibited. Because ligand stimulation of GPCRs also activates Src (17), we asked whether activation of GPCRs of the lysophosphatidic acid (LPA) family also triggered tyrosine phosphorylation of GIV. LPA stimulation resulted in the phosphorylation of wild-type GIV, but not the tyrosine phosphorylation–deficient mutant (Fig. 1F), indicating that Tyr1764 and Tyr1798 account for the observed tyrosine phosphorylation of GIV after LPA treatment. Phosphorylation of wild-type GIV was attenuated when cells were incubated with PP2 before LPA stimulation, indicating that ligand stimulation of the LPA receptor triggers tyrosine phosphorylation of GIV through Src family kinases. Collectively, these results demonstrate that both EGF and LPA trigger tyrosine phosphorylation of GIV: EGF triggers phosphorylation by EGFR, whereas LPA primarily uses non-receptor tyrosine kinases like the Src kinase family to phosphorylate GIV. We conclude that both receptor and non-receptor kinases can phosphorylate GIV in cells after ligand stimulation.

Tyrosine phosphorylation of GIV is required for enhancement of Akt phosphorylation, actin remodeling, and cell migration

To investigate whether tyrosine phosphorylation is required for GIV’s functions, such as enhancement of Akt activation, actin remodeling, and cell migration (18), we generated HeLa cell lines that were depleted of endogenous GIV by small interfering RNA (siRNA) (7) and that stably expressed an siRNA-resistant but otherwise wild-type form of GIV (HeLa–GIV-WT) or an siRNA-resistant form of GIV that could not be tyrosine phosphorylated (HeLa–GIV-YF) at an abundance of ~1.5- to 2-fold above that of endogenous GIV. In cells expressing the tyrosine phosphorylation GIV mutant and in GIV-depleted HeLa cells, phosphorylation of Akt was reduced compared to that in cells expressing wild-type GIV (Fig. 2A). Furthermore, cells expressing the tyrosine phosphorylation GIV mutant showed impaired formation of actin stress fibers and inefficient migration in scratch-wound assays (Fig. 2, B and C). Cells expressing a GIV mutant lacking a functional GEF motif (Phe1685→Ala) that cannot interact with or activate Gαi (7) fail to remodel actin, enhance Akt phosphorylation, or migrate after scratch-wounding (7). Akt phosphorylation was reduced to a greater extent in cells expressing the phosphorylation-deficient GIV mutant than in cells expressing the GIV mutant lacking the GEF motif (Fig. 2D). Regardless, expression of the tyrosine phosphorylation–deficient mutant (in HeLa–GIV-YF cells) or the mutant lacking a functional GEF motif (in HeLa–GIV-FA cells) did not rescue the functions of GIV when endogenous GIV was depleted (Fig. 2, B to D), indicating that both tyrosine phosphorylation of GIV and its GEF function are required for regulating Akt signaling and actin remodeling during cell migration.

Fig. 2

Tyrosine phosphorylation of GIV does not affect its ability to bind or activate Gαi but is required for phosphorylation of Akt, actin remodeling, and cell migration. (A) HeLa cells stably expressing control vector or siRNA-resistant GIV-WT–FLAG or GIV-YF–FLAG plasmids were transfected with GIV siRNA. Lysates were immunoblotted for GIV, phospho-Akt (pAkt), Gαi3, and tubulin. Phosphorylation of Akt (pAkt) was significantly reduced in GIV-depleted control cells (lane 2) and GIV-YF cells (lane 3) compared to GIV-WT cells (lane 1). P < 0.001 for both sets of comparisons; n = 3 experiments. (B) HeLa cells stably expressing vector (control), siRNA-resistant GIV-WT–FLAG, GIV-FA–FLAG (the GEF-deficient mutant), or GIV-YF–FLAG plasmids were treated with scrambled or GIV siRNA as indicated. Cells were costained with phalloidin–Texas Red (F-actin, red) and DAPI (DNA, blue). Depletion of GIV in control cells resulted in loss of stress fibers, which were restored by expression of siRNA-resistant GIV-WT–FLAG but not GIV-FA–FLAG or GIV-YF–FLAG. Scale bars, 10 μM. (C) Untransfected HeLa cells (Ctr, control), HeLa–GIV-WT, HeLa–GIV-FA, and HeLa–GIV-YF cells were transfected with scrambled or GIV siRNA. Results are shown as means ± SD of 8 to 12 randomly chosen fields from n = 3 experiments. P < 0.001 for either set of comparisons between Ctr and GIV-depleted cells and between GIV-WT–FLAG and GIV-YF–FLAG cells. (D) Control HeLa cells and HeLa cells stably expressing siRNA-resistant GIV-WT–FLAG, GIV-YF–FLAG, and GIV-FA–FLAG plasmids were transfected with scrambled and GIV siRNA and immunoblotted for GIV, pAkt, and tubulin. P < 0.001 for both comparisons between GIV-WT–FLAG and GIV-FA–FLAG and between GIV-WT–FLAG and GIV-YF–FLAG; n = 3 experiments. (E) Mock-treated His–GIV-CT and in vitro EGFR-phosphorylated His–GIV-CT were incubated with GST-Gαi3 or GST preloaded with GDP immobilized on glutathione beads. Bound proteins were analyzed by two-color immunoblotting (IB) for GIV-CT (red) and pTyr (green). (F) The amount of GTP hydrolyzed in 10 min by His-Gαi3 was determined in the presence of the indicated amounts of sham-treated and in vitro EGFR-phosphorylated His–GIV-CT.

The pTyrs of GIV function independently of its GEF motif

We sought to determine whether tyrosine phosphorylation affects the ability of GIV to bind and activate Gαi3. Both mock-treated and phosphorylated GIV C-terminal fragments bound Gαi3 to a similar extent in pull-down assays (Fig. 2E) and activated Gαi3, as assessed by the steady-state guanosine triphosphatase (GTPase) activity of the G protein, in a dose-dependent manner and with similar potencies (Fig. 2F). Furthermore, both GIV C-terminal fragments increased the GTPase activity of Gαi3 by ~1.7-fold over the basal activity at 0.6 μM, the highest concentration of GIV C-terminal protein tested. Thus, tyrosine phosphorylation of GIV does not affect the Gαi3-GIV interaction or the GEF activity of GIV toward Gαi3.

We next wanted to determine whether the GEF function of GIV was required for tyrosine phosphorylation of GIV. The GIV C-terminal fragment with the GEF motif mutation was phosphorylated to a similar extent as wild-type by EGFR in in vitro assays (fig. S4A) and in EGF-treated HeLa cells (fig. S4B), indicating that the GEF function was not required for tyrosine phosphorylation of GIV in cells. Thus, tyrosine phosphorylation and the GEF motif of GIV function independently of each other during cell migration.

Tyrosine-phosphorylated GIV binds the Src homology 2 domains of p85α

Tyrosine signaling pathways have been implicated in the activation of class 1 PI3Ks through direct binding to the Src homology 2 (SH2) domains of the p85α regulatory subunit of PI3Ks or indirect binding to other SH2 domain–containing proteins and adaptors (19, 20). To investigate whether tyrosine-phosphorylated GIV binds to SH2 domain–containing proteins, we performed pull-down assays using sham-treated or in vitro EGFR-phosphorylated His-GIV C-terminal fragments with a panel of SH2 adaptors. We used this approach because essential features of specific interactions between phosphoproteins and SH2 domain–containing proteins can be reconstituted with phosphopeptides and SH2 domain fragments (21). Phosphorylated, but not sham-treated, GIV C terminus bound to the C-terminal SH2 domain of p85α (p85α-CSH2) and, to a lesser extent, to the N-terminal SH2 domain of p85α (p85α-NSH2) (Fig. 3A) but not to other SH2 domains {c-Src, Gab1 [growth factor receptor–bound protein 2 (Grb2)–associated binding protein 1], or Grb2}. Phosphorylation of the GIV C terminus by EGFR or Src enabled binding to the C-terminal SH2 domain of p85α (Fig. 3B), suggesting that both receptor and non-receptor tyrosine kinases can generate pTyrs on the C terminus of GIV that are recognized by the SH2 domains of p85α. In addition, the C-terminal SH2 domain of p85α bound to more endogenous GIV from EGF-stimulated COS-7 cells than from serum-starved cells (fig. S5A). Furthermore, endogenously (Fig. 3, C and D) and exogenously expressed (fig. S5B) GIV coimmunoprecipitated with p85α after ligand stimulation of serum-starved cells. The formation of endogenous GIV-p85α complexes coincided with the timing of peak tyrosine phosphorylation of GIV and peak Akt activation, all of which occurred at ~5 min after ligand stimulation (Fig. 3C). Furthermore, the GIV-p85α association occurred after stimulation with any of the ligands tested (Fig. 3D and fig. S5B), suggesting that assembly of GIV-p85α complexes in cells can be triggered by multiple growth factors. However, the tyrosine phosphorylation–deficient GIV mutant coimmunoprecipitated with ligand-activated EGFR but not with p85α (Fig. 3E), thereby demonstrating that phosphorylation of Tyr1764 and Tyr1798 is required for the formation of GIV-p85α complexes in cells but not for the GIV-EGFR interaction. On the basis of our findings, we conclude that upon ligand stimulation GIV directly binds ligand-activated EGFR and enhances the recruitment of p85α to the activated receptor through binding of the SH2 domains of p85α to its C-terminally located pTyr.

Fig. 3

GIV interacts with p85α through its phosphorylated C terminus. (A) Mock-treated and in vitro EGFR-phosphorylated His–GIV-CT were incubated with the indicated GST-tagged SH2 domains or GST immobilized on glutathione beads. Bound His–GIV-CT was analyzed by immunoblotting (IB). (B) Mock-treated His–GIV-CT and in vitro EGFR- or Src-phosphorylated (pY-GIV-CT) His–GIV-CT were incubated with the GST-tagged C-terminal SH2 domain of p85α (GST-p85α-CSH2) or GST immobilized on glutathione beads. Bound His–GIV-CT was analyzed by immunoblotting (IB) with anti-His mAb. (C) Serum-starved HeLa cells were stimulated with EGF for the indicated time periods. GIV immunoprecipitates were immunoblotted for endogenous GIV, pTyr, and p85α. (D) Serum-starved HeLa cells were treated with the indicated growth factors. Immunoprecipitated complexes (top) were analyzed for GIV and p85α by immunoblotting (IB). (E) Serum-starved COS-7 cells expressing either FLAG-tagged WT (lanes 1 and 2) or the pTyr Y1764,Y1798F mutant (YF, lane 3) were stimulated with EGF. FLAG immune complexes (top) were immunoblotted for GIV-FLAG, ligand-activated EGFR (pTyr845 EGFR), and p85α.

Homology models reveal similarities between the GIV-p85α interface and canonical pTyr-SH2 interactions

To determine the relative contribution of Tyr1764 and Tyr1798 toward the observed interaction between the C terminus of GIV and the SH2 domains of p85α, we carried out pull-down assays on in vitro–phosphorylated wild-type or single tyrosine mutant forms of the GIV C-terminal fragment with the C-terminal SH2 domain of p85α. When EGFR was used to phosphorylate the GIV C-terminal fragments, the C-terminal SH2 domain of p85α bound to wild-type and the Y1798F mutant but not the Y1764F mutant (Fig. 4A). By contrast, when Src was used to phosphorylate the GIV C-terminal fragments, the C-terminal SH2 domain of p85α bound to wild-type and the Y1764F mutant but not the Y1798F mutant (Fig. 4A). Thus, EGFR triggers binding of GIV to p85α mostly through phosphorylation of Tyr1764, and Src does so mostly through phosphorylation of Tyr1798 (Fig. 1D). These results indicate that EGFR and Src create two distinct pTyr-binding sites on GIV for the C-terminal domain of p85α. In addition, both pTyrs also directly bound to the N-terminal SH2 domain of p85α (Fig. 4B), indicating that both N- and C-terminal SH2 domains of p85α have the ability to bind either of the two pTyrs on GIV.

Fig. 4

Structural basis for the GIV-p85α interaction. (A) His–GIV-CT WT (pY-WT) and single tyrosine mutants (pY1764F and pY1798F) were phosphorylated in vitro with recombinant EGFR or Src and used in pull-down assays with GST-p85α-CSH2 or GST immobilized on glutathione beads. Bound proteins were immunoblotted for His–GIV-CT. (B) In vitro–phosphorylated His–GIV-CT WT (pY-WT) and single tyrosine mutants (pY1764F and pY1798F) were used in pull-down assays with the GST-tagged N-terminal SH2 domain of p85α (GST-p85α-NSH2). (C) The sequence flanking Tyr1764 in GIV was aligned with phosphopeptide-binding sites on other p85α-interacting proteins. (D and E) Proposed structures of complexes between the C-terminal SH2 domain of p85α (D, pink) and the GIV-derived phosphopeptide EDTpY1764FISS (D, green) and between the N-terminal SH2 domain of p85α (E, green) and the GIV-derived phosphopeptide SNPpY1798ATLP (E, magenta) are shown. No steric clashes were observed in either model. Detailed views and descriptions of the predicted contacts are shown in fig. S6.

Next, we aligned GIV’s phosphopeptides (pY1764FISS and pY1798ATLP) with pTyr peptides from other proteins that bind to the C-terminal domain of p85α (Fig. 4C) and found that the GIV phosphopeptides do not resemble the canonical consensus sequence for peptides that bind the N- or C-terminal SH2 domains of p85α, pY[VMLI]XM (19). To gain insights into how these noncanonical phosphopeptides of GIV interact with the SH2 domains of p85α, we created the homology models (Fig. 4, D and E, and fig. S6) with the Internal Coordinate Mechanics (ICM) software (22, 23), using the crystal structures of the C-terminal SH2 domain of p85 in complex with pTyr751 peptide from PDGFRβ (24) or the N-terminal SH2 domain of p85 bound to c-Kit phosphotyrosyl peptide (25) as templates. pTyr1764 of GIV makes hydrogen bonds with residues of the C-terminal SH2 in a manner similar to that of the established crystal structure of this domain with pTyr751 of PDGFRβ. Similarly, pTyr1798 of GIV makes hydrogen bonds with residues of the N-terminal SH2 domain as seen in the resolved crystal structure of this domain complexed with phosphotyrosyl peptide of c-Kit. The side chains of both pTyrs of GIV make multiple hydrogen bonds with Arg631, Arg649, Ser651, and Ser652 of the C-terminal domain of p85α and with Arg340, Arg358, Ser361, Thr369, and Lys382 of the N-terminal domain of p85α, whereas the backbones of the phosphopeptides of GIV form hydrogen bonds with His669, His706, and Asn707 in the C-terminal domain of p85α and with Asn378, Leu380, and Asn417 in the N-terminal domain of p85α (Fig. 4, D and E, and fig. S6, A to D). Multiple polar and nonpolar contacts were also observed between the pTyr peptides of GIV and the SH2 domains of p85α. For example, Phe1765 of GIV-pY1764FISS bound a shallow hydrophobic pocket occupied by Val752 of PDGFRβ and Met724 of c-Kit in their respective complex structures with the C- and N-terminal SH2 domains of p85α, whereas Leu1801 of GIV pY1798ATLP occupies a deep cavity that binds Met754 of PDGFRβ and Met724 of c-Kit in their respective complex structures with the C- and N-terminal SH2 domains of p85α. These analyses suggest that the sequences flanking both pTyr peptides of GIV are equally compatible with direct and specific binding to either SH2 domain of p85α (Fig. 4, A and B).

The pTyrs of GIV couple PI3K to ligand-activated receptor tyrosine kinases

Because GIV directly binds ligand-activated EGFR (2) and p85α, we investigated whether GIV and, more specifically, its pTyr facilitate the recruitment of PI3K to activated EGFR. In HeLa cells expressing wild-type GIV, EGFR transiently and maximally associated with p85α at 5 min after ligand stimulation, whereas in cells expressing the tyrosine phosphorylation–deficient GIV mutant, p85α recruitment was reduced (Fig. 5A). Thus, tyrosine phosphorylation of GIV is critical for effective formation or stabilization of the p85α-EGFR complex. Similar results were obtained with exogenously expressed insulin receptor (InsR) (Fig. 5B), suggesting that tyrosine phosphorylation of GIV may also be critical for effective formation or stabilization of a p85α-InsR complex. These results suggest that interactions between receptor tyrosine kinases and p85α in cells are enhanced by GIV through its C-terminal pTyr.

Fig. 5

Tyrosine phosphorylation of GIV stabilizes receptor-p85α complexes and increases PI3K activity. (A) EGFR immunoprecipitates from serum-starved or EGF-stimulated HeLa–GIV-WT and HeLa–GIV-YF cells were immunoblotted for total (t-EGFR) and activated (pY-EGFR) EGFR and p85α. (B) HA immunoprecipitates (containing HA-tagged InsR; InsR-HA) from serum-starved and insulin-treated HeLa–GIV-WT and HeLa–GIV-YF cells were immunoblotted for HA (to detect InsR-HA), pTyr, and p85α. (C) COS-7 cells expressing p85α-HA and either WT or pTyr mutant (YF) of GIV-FLAG were maintained in 2% fetal bovine serum (FBS) for 24 hours. PI3K assays (left) were carried out with immunoprecipitated p85α-HA, phosphatidylinositol, and [γ-32P]ATP. Lysates and HA immunoprecipitates (containing p85α-HA) were immunoblotted for the indicated proteins (right). (D) HeLa cells stably expressing either siRNA-resistant GIV-WT, pTyr mutant GIV (GIV-YF), or vector control were transfected with scrambled or GIV siRNA and then with Akt-PH-GFP plasmid. Cells were serum-starved or treated with EGF and then stained for GFP (yellow) and with DAPI (blue). Scale bars, 10 μM. (E) Lysates (left) from 21T series of cells (16N, NT, and MT2) were immunoblotted for GIV, p85α, pAkt, Gαi3, and tubulin. GIV immunoprecipitates from lysates (normalized for equal abundance of GIV protein) were analyzed by two-color immunoblotting (IB) for GIV (red), p85α, and pTyr (green).

Tyrosine-phosphorylated GIV activates PI3K at the plasma membrane

Binding of pTyr to both SH2 domains of the regulatory p85α subunit activates the catalytic p110 subunit of PI3K (19, 26), which triggers the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] (2731). Because pTyrs of GIV directly bind to the SH2 domains of p85α, this interaction would be expected to activate PI3K. We carried out in vitro PI3K assays (32) with purified phosphoinositides and immunoprecipitated p85α from cells expressing vector, wild-type GIV, or the tyrosine phosphorylation–deficient form of GIV. The activity of PI3K, as determined by the extent of production of phosphatidylinositol 3-phosphate, was low in cells expressing vector or the tyrosine phosphorylation–deficient form of GIV and was higher in cells expressing wild-type GIV (Fig. 5C), indicating that phosphorylation of Tyr1764 and Tyr1798 in GIV enhances PI3K activity in cells. To determine whether this activation of PI3K occurred at the plasma membrane and whether the abundance of PIP3 was increased, we expressed the green fluorescent protein (GFP)–tagged pleckstrin homology (PH) domain of Akt (Akt-PH-GFP) (Fig. 5D), which recognizes PIP3 (3335), and monitored its EGF-dependent recruitment to the plasma membrane. In serum-starved HeLa control cells, the Akt-based PIP3 reporter was mostly cytosolic but was recruited to the plasma membrane within 10 min after EGF stimulation, indicating activation of PI3K and generation of PIP3 at the plasma membrane after ligand stimulation. Cells expressing wild-type GIV showed a similar pattern of robust EGF-triggered recruitment of the Akt-based PIP3 reporter to the plasma membrane. By contrast, in cells expressing the tyrosine phosphorylation–deficient form of GIV, the Akt-based PIP3 reporter remained cytosolic before and after EGF treatment, indicating that the abundance of PIP3 at the plasma membrane does not change in these cells after ligand stimulation. To circumvent the possibility that altered localization of Akt in cells expressing the tyrosine phosphorylation–deficient form of GIV could be due to changes in interaction between GIV and Akt (1, 5), we performed similar assays with a second validated reporter for PIP3 that contains the PH domain of Bruton’s tyrosine kinase (36) (Btk-PH-GFP) (fig. S7). Findings with the Btk-based PIP3 reporter were similar to those with the Akt-based PIP3 reporter, demonstrating that EGF-triggered production of PIP3 is impaired in cells expressing the tyrosine phosphorylation–deficient form of GIV. We conclude that ligand-stimulated activation of PI3K and production of PIP3 at the plasma membrane requires tyrosine phosphorylation of GIV.

Tyrosine phosphorylation of GIV and its association with p85α increase during metastatic progression of a breast carcinoma

We previously showed that the abundance of GIV protein and mRNA increases during metastatic progression in colorectal and breast carcinomas (6), an increase that coincides with increased activity of the PI3K-Akt pathway in tumor cells (37, 38). Using the 21T series of human mammary cells (16N, NT, and MT2), we investigated the extent of tyrosine phosphorylation of GIV and its association with p85α during metastatic progression (2). These cells were derived by successive biopsies from a single patient with breast cancer: 16N is from the normal breast, NT from the primary tumor (invasive ductal carcinoma), and MT2 from the metastatic pleural effusions. Consistent with our previous work (2, 9), the abundance of full-length GIV and extent of Akt phosphorylation were lowest in 16N, intermediate in NT, and highest in MT2 (Fig. 5E). Tyrosine phosphorylation of GIV and the amount of p85α that coimmunoprecipitated with GIV were lowest in 16N, intermediate in NT, and highest in MT2 cells (Fig. 5E). These results suggest that tyrosine phosphorylation of GIV and its association with p85α may increase during metastatic progression of breast carcinoma and that GIV-dependent PI3K activation may play a role during tumor invasion.

Tyrosine phosphorylation of GIV is required for Akt-dependent phosphorylation of GIV at Ser1416

The phosphorylation of GIV at Ser1416 by Akt has been attributed to multiple biological functions of GIV, including cancer invasion and metastasis, neoangiogenesis, control of cell size during development, neuronal migration, and vascular repair after injury (14, 6, 8, 9, 3942). Thus, we investigated the relationship between tyrosine phosphorylation and Akt-dependent serine phosphorylation of GIV. Phosphorylation of wild-type GIV at Ser1416 was higher than that of the tyrosine phosphorylation–deficient mutant (Fig. 6A), suggesting that tyrosine phosphorylation of GIV is a prerequisite for efficient Akt-dependent serine phosphorylation of GIV. The serine phosphorylation–deficient Ser1416→Ala (S1416A) and the serine phosphorylation–mimicking Ser1416→Asp (S1416D) mutants of GIV were tyrosine phosphorylated to a similar extent as wild-type GIV (Fig. 6B), indicating that the phosphorylation status of Ser1416 does not affect tyrosine phosphorylation of GIV. Together, these findings suggest that tyrosine phosphorylation of GIV occurs upstream of and regulates the subsequent step of Akt-dependent phosphorylation of GIV at Ser1416.

Fig. 6

The dual role of GIV as an enhancer of and as a substrate for Akt kinase. (A) COS-7 cells expressing vector, GIV-WT–FLAG, or GIV-YF-FLAG were maintained in the presence of 2% FBS. FLAG immunoprecipitates were analyzed by two-color immunoblotting (IB) for FLAG (green; to detect GIV) and pSer1416 GIV (red). (B) COS-7 cells expressing WT (GIV-WT–FLAG), phosphoserine mutants of GIV (GIV-S1416A-FLAG and GIV-S1416D), or vector control were serum-starved or stimulated with EGF. FLAG immunoprecipitates were analyzed by two-color immunoblotting (IB) for GIV (red) and pTyr (green). (C) Upon ligand stimulation, EGFR is activated and autophosphorylated. GIV is recruited to the autophosphorylated cytoplasmic tails of activated receptors. Activated receptor and non-receptor tyrosine kinases (such as c-Src) phosphorylate GIV at two tyrosine residues, which are direct binding sites for the SH2 domains of the regulatory p85α subunit of PI3K. We propose that GIV stabilizes the receptor-p85α complexes, triggers the activation of PI3K, and augments the production of PIP3 at the plasma membrane. (D) Working model for how GIV enhances class 1 PI3K activity. The pTyr of GIV directly interact with p85α and activate class 1A PI3K (solid, left arrow). Akt-dependent phosphorylation of GIV at Ser1416 requires GIV-dependent activation of the PI3K-Akt pathway. This implies that tyrosine phosphorylation of GIV occurs upstream of Akt-mediated phosphorylation of GIV; the latter event may set up a feedback loop (interrupted, left arrow). The GEF motif of GIV activates Gi and releases free Gβγ subunits, which in turn directly interact and activate the catalytic p110γ subunits of class 1B PI3Ks (solid, right arrow) (62). The Gβγ pathway can also bind and activate the catalytic p110β subunits of class 1A PI3Ks (interrupted, right arrow) (61). Thus, the GIV-mediated direct and indirect enhancement of class 1 PI3K activity may synergistically amplify PI3K activity at the plasma membrane.

Our results demonstrate that GIV is a substrate of multiple tyrosine kinases and that the pTyrs of GIV directly bind to and activate Akt and PI3K at the plasma membrane (Fig. 6C). Activated Akt subsequently phosphorylates GIV and triggers cell migration (5, 7, 8). On the basis of these findings, we propose that multiple receptors enhance PI3K activity and trigger cell migration through a common tyrosine phosphoprotein, GIV.

Discussion

Tyrosine-phosphorylated GIV serves as a common platform to enhance PI3K activity during cell migration

Here, we describe GIV, a non-receptor GEF for Gαi3, as a key molecule in the tyrosine signaling pathway. We demonstrate that GIV is a common substrate for multiple receptor and non-receptor tyrosine kinases and identify the sites of phosphorylation as Tyr1764 and Tyr1798 in its C terminus. These pTyrs are required for GIV to bind to p85α in vitro and for the ligand-dependent formation of GIV-p85α complexes in cells. In HeLa cells expressing wild-type GIV, in which Tyr1764 and Tyr1798 undergo ligand-dependent phosphorylation, PI3K is activated to generate PIP3 at the plasma membrane, activation of Akt is enhanced, actin is remodeled, and cell migration is triggered. By contrast, HeLa cells expressing a GIV mutant in which both tyrosines are replaced by phenylalanines show defective ligand-dependent activation of PI3K, lack of enhanced Akt activation, absence of actin remodeling, and lack of migration. We conclude that GIV enhances ligand-stimulated PI3K activity through pTyr in its C terminus.

We also provide the mechanism for GIV-dependent enhancement of PI3K activity by two diverse classes of chemotactic receptors: growth factor receptor tyrosine kinases and GPCRs. We found that GIV was tyrosine phosphorylated after cells are stimulated either with growth factors like EGF and insulin or with a GPCR ligand like LPA. Upon EGF stimulation, GIV was phosphorylated at two particular tyrosine residues by activated EGFR, whereas upon LPA stimulation GIV was phosphorylated at these same residues primarily by Src family kinases. Tyrosine-phosphorylated GIV bound the regulatory p85α subunit of PI3K. Thus, tyrosine phosphorylation of GIV and the formation of GIV-p85α complexes are triggered by multiple receptors that require GIV to enhance Akt signals (2, 68, 43). We conclude that the tyrosine kinase–phospho-GIV–p85α axis serves as a common pathway initiated by multiple receptors to enhance PI3K activity during cell migration.

Previous work has established that migrating cells display a steep PI3K-Akt signaling gradient that is restricted to the leading edge (34, 44). However, the uniformity of distribution of chemotactic receptors (receptor tyrosine kinases and GPCRs) (4547) and shallow anterior-posterior gradient of Gβ subunits (48, 49) have failed to account for the observed signaling gradient. Because GIV localizes to the plasma membrane at the leading edge of migrating cells (1, 3, 4, 8), we propose that tyrosine-phosphorylated GIV could contribute to the generation and/or maintenance of the steep PI3K signaling gradient observed at the leading edge of migrating cells.

The pTyrs of GIV directly bind p85α

We also provide the structural basis for GIV’s association with the p85α regulatory domain of PI3K. Upon phosphorylation, GIV directly interacts with p85α through its two C-terminally located pTyrs, Tyr1764 and Tyr1798. Both tyrosines are substrates for EGFR and Src, and tyrosine-phosphorylated GIV interacted with both N- and C-terminal SH2 domains of p85α. The ability of the pTyr of GIV to bind both SH2 domains of p85α is in accordance with the structural similarity between the N- and the C-terminal SH2 domains of p85α (21) and the ability of both domains to recognize and bind similar pTyr sequences (21). The sequences flanking these two tyrosine residues in GIV differ from the canonical p85α-binding YXXMX consensus (50) and instead resemble other noncanonical p85α-binding sequences. The pY1798ATLP peptide shares homology with the pY343LVL peptide from erythropoietin (EPO) receptor (50), whereas the pY1764FISS peptide shares homology with the pYEPTG peptide from Syk (51) or the pYVNTT peptide in Tie2 (33), which bind the C-terminal SH2 domain of p85α with high affinity and specificity. Thus, the pY1764FISS motif of GIV and its counterparts in Syk and Tie2 are a noncanonical class of peptides with a pYXX[ST] consensus.

We noted that the spacing of GIV’s pTyr and the distance between the two SH2 domains of p85α (52) are compatible with the possibility that the tandem C-terminal pTyr of GIV simultaneously occupy the tandem SH2 domains of p85α. Previous work has established that such double occupancy of p85α-SH2 domains in tandem is required for full activation of the catalytic p110 subunit, presumably through one of the two proposed mechanisms: by triggering allosteric conformational changes (53) or by promoting kinase oligomerization (53). Compared to single occupancy, in-tandem double occupancies in the tyrosine-SH2 signaling pathway confer substantially higher affinity and enhanced biological specificity (52, 54). Whether the biological specificity and potency of PI3K activation by the pTyr of GIV that we observe stems from similar in-tandem interactions remain to be investigated. We conclude that binding of tyrosine-phosphorylated GIV to the SH2 domains of p85α provides a mechanism by which signals could be transmitted directly from activated tyrosine kinases to PI3K.

GIV enhances the formation of receptor-PI3K complexes

We found that tyrosine-phosphorylated GIV facilitated recruitment of p85α to activated EGFR and InsR. Wild-type GIV bound ligand-activated receptor tyrosine kinases and was phosphorylated on the C-terminal tyrosine residues that directly bound the p85α subunit of PI3K. In the case of the GIV mutant lacking the C-terminal tyrosine phosphorylation sites, binding to ligand-activated receptors was intact, but phosphorylation could take place and p85α recruitment to receptor was reduced (perhaps as a result of a dominant-negative effect of this mutant obscuring other sites for adaptor binding). Previous work has established that the SH2 domains of p85α bind with high affinity to autophosphorylation sites on the cytoplasmic tails of certain receptor tyrosine kinases and in doing so are primarily responsible for the binding of PI3K to activated growth factor receptors (55). To date, such high-affinity, direct p85α-binding sites have been described only among receptors of the PDGFR family, such as PDGFR, Kit, and CSF-1 (colony-stimulating factor 1) (56). Although several receptors [VEGFR, InsRβ, EGFR, and IGFR (IGF receptor)] interact with the SH2 domains of p85α upon autophosphorylation, these interactions either are weaker than that observed between PDGFR and p85α [as in the case of EGFR (56) and IGFR (57)] or are yet to be demonstrated to be direct [as in the case of VEGFR (58) and InsR (59)]. Although other adaptors—Grb2 and Gab1 in the case of EGFR and IRS1 (insulin receptor substrate 1) in the case of InsR—can indirectly couple p85α to receptor tyrosine kinases, a specific biological role for PI3K activation through these linkers remains to be established (60). We propose that simultaneously binding of GIV to receptors and p85α may stabilize receptor tyrosine kinase–PI3K complexes in cells and may provide a mechanism by which PI3K signals are amplified directly by GIV at the immediate post-receptor level during cell migration.

The GEF and pTyr motifs of GIV enhance activation of distinct subclasses of PI3Ks

Our findings identify tyrosine phosphorylation of the C terminus of GIV as a key underlying event that triggers enhancement of PI3K activation through GIV. We previously demonstrated an obligate requirement also of the GEF motif of GIV for enhancement of PI3K-Akt signaling (7). The GEF motif of GIV is separated from the critical pTyr motifs by ~80 to 110 amino acids. Here, we provide evidence that these two motifs function independently of each other. In addition, we define the direct link between GIV’s pTyr and p85α, which is a class 1A PI3K (19). We previously defined the link between activation of Gi by the GEF motif of GIV and activation of PI3K (7): GIV activates G protein and releases “free” Gβγ subunits (7), which in turn bind and activate p110γ (PI3K) (61, 62), a class 1B PI3K, and to some extent p110β, a class 1A PI3K (63). Thus, we propose (Fig. 6D) that enhancement of cellular PI3K activity by GIV requires simultaneous activation of two parallel pathways: (i) GIV binds p85α regulatory subunits of class 1A PI3K, and (ii) GIV activates Gi and releases free Gβγ, which then binds p110 catalytic subunits of class 1A and 1B PI3Ks (1, 3, 4). Although our results demonstrate that both pathways are required for full activation of the GIV-PI3K-Akt axis, the relative contributions of each pathway remain unknown and may vary depending on whether growth factor receptors or GPCRs are stimulated.

We also provide evidence that the ability of GIV to enhance Akt through the GIV-PI3K-Akt axis increases Akt-dependent phosphorylation of GIV at Ser1416, a key phosphorylation event that activates or triggers many biological functions of GIV (1, 3, 4, 39, 40, 42). Although tyrosine phosphorylation of GIV was a prerequisite for efficient phosphorylation of GIV at Ser1416 by Akt, presumably through enhancement of Akt activity, phosphorylation at Ser1416 did not alter the tyrosine phosphorylation of GIV. These findings suggest a distinct hierarchy in signal transduction: The GIV-PI3K-Akt axis operates upstream at an immediate post-receptor level to amplify incoming PI3K-Akt signals and possibly receives regulatory feedback from the downstream Akt-GIV axis wherein Akt phosphorylates and triggers GIV’s biological functions (Fig. 6D). Such a hierarchy or feedback loop may not be universally operational, because GIV may still be phosphorylated by Akt in certain cell lines, irrespective of whether it plays a major role in enhancement of Akt activity in those cells (40). Our results provide insights into how GIV’s function both as an enhancer of Akt activity and as a substrate of Akt might be intertwined.

Tyrosine phosphorylation of GIV and its association with p85α may increase during cancer progression

The activation of the PI3K-Akt pathway is frequently increased during cancer invasion (38, 64), and progressive enhancement of PI3K-Akt coupled to efficient cell migration is a hallmark of high metastatic potential (38). We previously reported (2, 8, 9) that higher metastatic potential is associated with higher abundance of GIV mRNA and protein in breast and colon carcinomas, and others (3, 4) have demonstrated that GIV is required for tumor cell invasion and VEGF-mediated neoangiogenesis during cancer metastasis. Here, we show that tyrosine phosphorylation of GIV and the abundance of GIV-p85α complexes were increased in a breast carcinoma cell during metastatic progression. These changes in GIV’s properties among 21T cells coincide with the previously observed (38) increases in the extent of Akt phosphorylation, efficacy of migration, and invasiveness in mouse models of cancer metastasis. It is possible that the observed increases in tyrosine phosphorylation of GIV and the abundance of GIV-p85α complexes in breast carcinoma cells lead to activation of the GIV-PI3K-Akt axis during cancer metastasis. Finally, this axis is assembled exclusively in cancer cells and tumors with high invasiveness because GIV’s C-terminally located pTyrs are excluded in an alternative spliced, truncated GIVΔCT variant we reported (2) to exist in poorly invasive breast and colon cancer cell lines and noninvasive colorectal carcinomas. Our findings suggest that the GIV-PI3K-Akt axis may substantially contribute toward increased activation of the PI3K-Akt pathway observed during cancer invasion. Whether abolishing phosphorylation of the C-terminal tyrosine residues of GIV or disruption of the GIV-p85α interface are feasible therapeutic strategies to halt cancer progression or whether detection of pTyr-GIV can be a prognosticator of survival remains to be investigated.

In conclusion, we have demonstrated that GIV is a central hub for PI3K activation within tyrosine-based signaling networks, upon which multiple receptors converge to enhance Akt activity. The mechanistic and structural insights gained herein not only characterize a new pharmacological target for the regulation of PI3K activity but also define the previously elusive links among receptors, GIV, and its role in enhancing Akt activation during cell migration.

Materials and Methods

Reagents and antibodies

Unless otherwise indicated, all reagents were of analytical grade and obtained from Sigma-Aldrich. Cell culture media were purchased from Invitrogen. EGF and insulin were obtained from Invitrogen and Novagen, respectively. Recombinant EGFR, VEGFR, and PDGFRβ were purchased from Cell Signaling Technology. PP2 was obtained from Calbiochem. Silencer Negative Control scrambled (Scr) siRNA and Gαi3 siRNA (8) were purchased from Ambion and Santa Cruz Biotechnology, respectively, whereas GIV siRNA (2, 7, 8) was custom-ordered from Dharmacon. Antibodies against GIV that were used in this work include rabbit serum and affinity-purified anti-GIV coiled-coil immunoglobulin G (IgG) (GIV-ccAb; for immunoblotting only) raised against the coiled-coil domain of GIV (2, 7, 8) and affinity-purified anti-Girdin C terminus (GIV-CTAb; for immunoprecipitation) raised against the last 19 amino acids of the C terminus of GIV (IBL-America and Santa Cruz Biotechnology). Total EGFR was visualized by immunofluorescence with monoclonal antibody (mAb) #225 raised against the ectodomain [gift from G. Gill, University of California, San Diego (UCSD) (65)] or anti-EGFR polyclonal antibody (Santa Cruz Biotechnology). Mouse mAbs against pTyr (BD Biosciences, cat. #610000), FLAG (Sigma, for immunoprecipitation), polyhistidine (Sigma), GFP (Living Colors, Invitrogen), hemagglutinin (HA) (Covance), and tubulin (Sigma) were purchased from commercial sources. Rabbit polyclonal antibodies against FLAG (Invitrogen; for immunoblotting), p85α (Millipore Inc.), Gαi3 (M-14; Santa Cruz Biotechnology), phospho-Akt S473 (Cell Signaling Technology), pan-Gβ (Santa Cruz Biotechnology), and phospho-ERK1/2 (extracellular signal–regulated kinases 1 and 2) (Cell Signaling Technology) were obtained commercially. Anti-mouse and anti-rabbit Alexa Fluor 594– and Alexa Fluor 488–coupled goat secondary antibodies for immunofluorescence were purchased from Invitrogen. Goat anti-rabbit and goat anti-mouse Alexa Fluor 680 or IRDye 800 F(ab′)2 for immunoblotting were from LI-COR Biosciences. Control mouse and rabbit IgGs for immunoprecipitations were purchased from Bio-Rad and Sigma, respectively.

Plasmid constructs and mutagenesis

Cloning of Gαi3 and GIV into pGEX-4T-1 or pET28b was described previously (7). Glutathione S-transferase (GST)–EGFR-T (amino acids 1046 to 1210) was cloned into pGEX-4T-1 on the basis of the reported sequence (NM_005228) as described previously (7). For mammalian expression, C-terminal FLAG-tagged GIV was generated by cloning GIV into p3XFLAG–CMV-14 between Not I and Bam H1. RNA interference (RNAi)–resistant GIV was generated by silent mutations as described previously (7). FLAG-GIV and His–GIV-CT phosphomutants (Y1764F, Y1798F, and Y1764,1798F) were generated by site-directed mutagenesis with a QuikChange kit (Stratagene) as per the manufacturer’s protocols. GST-Src (amino acids 1 to 257, accession number NM_001025395) was cloned into pGEX-4T-1 between Eco RI and Bam H1. C-terminal HA-tagged c-Src for mammalian expression was generated by cloning the entire coding sequence into pcDNA 3.1 between Xho I and Eco RI. The following plasmids and constructs were gifts from other investigators: untagged EGFR (66) and GST–TrkA-CT (amino acids 448 to 552) construct encoding the 75–amino acid, juxtamembrane region of rat TrkA from M. G. Farquhar (UCSD) (67); GST-p85 N- and C-SH2 constructs from R. Rajala (University of Okalahoma Health Sciences Center, Oklahoma City, OK) (59); GST-Grb2 and Gab1 from M. Holgado-Madruga (Stanford, CA) (68); GFP-Akt-PH from R. Tsien (UCSD); GFP-Btk-PH from S. J. Field (UCSD); GST–PLC-γ1 (phospholipase C–γ1) N- and C-SH2 from T. Pawson (Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada) (55); p85α-HA from H. Band (Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE) (69). All constructs were checked by DNA sequencing.

Protein expression and purification

GST, GST-Gαi3, GST–EGFR-T (amino acids 1046 to 1210), GST–TrkA-CT (amino acids 448 to 552), the various GST-SH2 adaptors (p85-NSH2, p85-CSH2, Src-SH2, Gab1, Grb2), His-Gαi3, His–GIV-CT WT (amino acids 1660 to 1870), and phosphomutant (Y1764F, Y1798F, and Y1764,1798F) constructs were expressed and purified from Escherichia coli strain BL21(DE3) (Invitrogen) as described previously (7, 70). Briefly, cultures of transformed bacteria were induced overnight at 25°C with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and bacterial pellet from 1 liter of culture was resuspended in 10 ml of GST lysis buffer [25 mM tris-HCl (pH 7.5), 20 mM NaCl, 1 mM EDTA, 20% (v/v) glycerol, 1% (v/v) Triton X-100, and 2× protease inhibitor cocktail (Complete EDTA-free, Roche Diagnostics)] or His lysis buffer [50 mM NaH2PO4 (pH 7.4), 300 mM NaCl, 10 mM imidazole, 1% (v/v) Triton X-100, and 2× protease inhibitor cocktail (Complete EDTA-free, Roche Diagnostics)] for GST- or His-fused proteins, respectively. After sonication (4× 20 s, 1 min between cycles), lysates were centrifuged at 12,000g at 4°C for 20 min. Solubilized proteins were affinity-purified on glutathione-Sepharose 4B beads (GE Healthcare) or HisPur Cobalt Resin (Pierce). Proteins were eluted, dialyzed overnight against phosphate-buffered saline (PBS), and stored at −80°C. His-Gαi3 was buffer-exchanged into G protein storage buffer [20 mM tris-HCl (pH 7.4), 200 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 10 μM guanosine diphosphate (GDP), and 5% (v/v) glycerol] before storage at −80°C.

In vitro and cellular phosphorylation assays

In vitro kinase assays were performed with bacterially expressed His [6 × His (hexahistidine)]–tagged GIV-CT (His–GIV-CT, amino acids 1660 to 1870) proteins (~10 to 15 μg per reaction) and recombinant kinases that were obtained commercially (EGFR, Millipore Inc., and Cell Signaling Technology; c-Src, VEGFR2, and PDGFRβ, Cell Signaling Technology) or expressed in bacteria (GST-TrkA kinase domain). Reactions were started by adding 200 to 1000 μM adenosine 5′-triphosphate (ATP) and carried out at 25°C for 60 min in tyrosine kinase buffer [60 mM Hepes (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, 3 μM Na3OV4]. Reactions were stopped by addition of Laemmli sample buffer and boiling at 100°C. For cellular phosphorylation assays on endogenous GIV (Fig. 1B and fig. S1), HeLa cells were serum-starved for 12 to 16 hours and preincubated with 100 μM sodium orthovanadate and, where indicated, the Src inhibitor PP2 (250 nM) for 1 hour before EGF (50 nM), insulin (100 nM), or LPA (20 μM) stimulation. Reactions were stopped with PBS chilled at 4°C, supplemented with 200 μM sodium orthovanadate, and immediately scraped and lysed for immunoprecipitation. For cellular phosphorylation assays using overexpressed GIV, FLAG-tagged GIV was coexpressed with untagged EGFR (Fig. 1E) or HA-tagged Src (Fig. 1F), and at 32 hours after transfection, cells were processed as described above. Tyrosine phosphorylation was analyzed by immunoblotting with anti-pTyr mAb (BD Biosciences).

Phosphopeptide enrichment and liquid chromatography–tandem MS analysis

In vitro–phosphorylated His–GIV-CT protein was resuspended, reduced with tris(2-carboxyethyl)phosphine (TCEP), and carboxymethylated with iodoacetamide before digestion with trypsin. Samples were then processed as described previously (71), and the phosphopeptides were enriched with TiO2 (72) before their separation and analysis by nanoflow high-pressure liquid chromatography (HPLC) coupled with tandem MS (LC-MS/MS) by means of a QSTAR-Elite hybrid mass spectrometer (AB Sciex) (73). The collected data were searched with Protein Pilot 2.0 (AB Sciex) and MASCOT (Matrix Science) for sequence identifications.

Cell culture, transfection, and lysis

Unless mentioned otherwise, cell lines used in this work were cultured according to the American Type Culture Collection guidelines. Transfection was carried out with GeneJuice (Novagen) for DNA plasmids or Oligofectamine (Invitrogen) for siRNA oligos according to the manufacturer’s protocols, and stable cell lines were selected as mentioned previously (2, 7) with the neomycin analog G418 (Cellgro). HeLa cell lines stably expressing GIV-WT (HeLa–GIV-WT), GIV-F1685A mutant (HeLa–GIV-FA), and GIV-Y1764,1798F mutant (HeLa–GIV-YF) were generated and maintained in the presence of G418 (500 μg/ml) as previously described. Clones were chosen for each construct that had relatively low abundance of exogenously expressed GIV (about two times the abundance of endogenous GIV). For each construct, similar results were obtained from two separate clones. The 21T breast cell lines (16N, NT, and MT2) were obtained from A. B. Pardee (Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA) and maintained as described previously (37, 38).

Lysates for immunoprecipitation or pull-down assays were prepared by resuspending cells in lysis buffer [20 mM Hepes (pH 7.2), 5 mM magnesium acetate, 125 mM potassium acetate, 0.4% Triton X-100, 1 mM DTT, supplemented with sodium orthovanadate (500 μM), phosphatase (Sigma), and protease (Roche) inhibitor cocktails], after which they were passed through a 30-gauge needle at 4°C and cleared (10,000 to 14,000g for 10 min) before use in subsequent experiments.

Steady-state GTPase assay

These assays were done as described previously (2, 7, 8). Briefly, 100 nM His-Gαi3 was preincubated with different concentrations of sham-treated or in vitro EGFR-phosphorylated His–GIV-CT (amino acids 1660 to 1870) for 15 min at 30°C in assay buffer [20 mM Na Hepes (pH 8), 100 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 1 mM DTT, 0.05% (w/v) C12E10]. GTPase reactions were initiated at 30°C by an equal volume of assay buffer containing 1 μM [γ-32P]GTP (guanosine 5′-triphosphate) (~50 cpm/fmol). Duplicate aliquots (50 μl) were removed at 10 min, and reactions were stopped with 950 μl of ice-cold 5% (w/v) activated charcoal in 20 mM H3PO4 (pH 3). Samples were then centrifuged for 10 min at 10,000g, and 500 μl of the resultant supernatant was scintillation-counted to quantify released [32P]Pi. To determine the specific Pi produced, we subtracted the background [32P]Pi detected at 10 min in the absence of G protein from each reaction. The results were expressed as absolute values of Pi produced.

Immunofluorescence

Cells were fixed at room temperature with 3% paraformaldehyde for 20 to 25 min, permeabilized (0.2% Triton X-100) for 45 min, and incubated for 1 hour each with primary and then secondary antibodies as described previously (8). Antibody dilutions were given as follows: mAb GFP, 1:500; secondary goat anti-rabbit (594) and goat anti-mouse (488) Alexa Fluor–conjugated antibodies, 1:500; and 4′,6-diamidino-2-phenylindole (DAPI), 1:2000 (Molecular Probes). Samples were examined with a Zeiss Axiophot microscope (Carl Zeiss Inc.) with a 63× aperture [Zeiss Plan Neofluar, 1.30 numerical aperture (NA)], and images were collected with an ORCA-ER camera (Hamamatsu) and Volocity Software. All individual images were processed with ImageJ software (National Institutes of Health) and assembled for presentation with Photoshop and Illustrator software (both Adobe).

GST pull-down and immunoprecipitation assays

These assays were carried out as previously described (24) with minor modifications. Purified GST-fused proteins (15 to 35 μg) or GST alone (30 to 45 μg) were immobilized on glutathione S-Sepharose beads (GE Healthcare) and incubated for 4 hours at 4°C in binding buffer [50 mM tris-HCl (pH 7.4), 100 mM NaCl, 0.4% (v/v) NP-40, 10 mM MgCl2, 5 mM EDTA, 2 mM DTT, and 2 mM sodium orthovanadate] containing sham-treated or in vitro–phosphorylated His–GIV-CT (amino acids 1660 to 1870). After a 4-hour incubation at 4°C, the beads were washed [4.3 mM Na2HPO4, 1.4 mM KH2PO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl, 0.1% (v/v) Tween 20, 10 mM MgCl2, 5 mM EDTA, 2 mM DTT, and 30 and 2 mM sodium orthovanadate] and bound proteins were eluted in sample buffer for SDS–polyacrylamide gel electrophoresis (SDS-PAGE). When GST-Gαi3 was used in these assays, both binding and wash buffers were supplemented with 30 μM GDP. Where indicated, His–GIV-CT was phosphorylated in vitro with recombinant EGFR (Invitrogen) before its use in pull-down assays.

For immunoprecipitations, cell lysates (~1 to 2 mg of protein) were incubated for 4 hours at 4°C with either 2 μg of anti-FLAG mAb for immunoprecipitation of GIV-FLAG, anti–GIV-CT (Girdin-T13 Ab, Santa Cruz Biotechnology) for endogenous GIV, anti-HA mAb (Covance) for immunoprecipitation of HA-tagged InsR, anti-EGFR #225 mAb (65) for immunoprecipitation of endogenous EGFR, or their respective pre-immune control IgGs where indicated. Protein A (for GIV-CTAb) or G (for all other mAbs) agarose beads (GE Healthcare) were added and incubated at 4°C for an additional 60 min. Beads were washed and then either resuspended or boiled in SDS sample buffer. Buffers were supplemented with 1 mM sodium orthovanadate for all steps of the assay.

Molecular modeling of the GIV-p85 interface

The initial coordinates for C- and N-terminal SH2 domains of p85α were taken from their crystal structures bound to the PDGFRβ peptide, pY751VPML (24), and c-Kit phosphotyrosyl peptide (25), respectively. Structural models of GIV’s pTyr peptides, EDTpY1764FISS and SNPpY1798ATLP, were initially generated with ideal covalent geometry. Backbone atoms of the peptides and heavy atoms of the phosphorylated tyrosine side chain were tethered to their respective counterparts in the PDGFRβ or c-Kit peptide templates by soft harmonic restraints and subjected to several rounds of Monte Carlo optimization with decreasing tether weight. During the optimization, the backbone conformation of the SH2 domain was held constant, and torsional angles controlling the side chains of both molecules and the backbone of the GIV pTyr peptide were fully sampled.

PI3K assay

The method we used here is largely adapted from published protocols (2731) with minor modifications. COS-7 cells plated at 80 to 85% confluency were cotransfected with p85α-HA and either wild-type or YF mutant of GIV-FLAG. Vector-transfected COS-7 cells were used as controls. Forty-eight hours after transfection, cells were lysed in the lysis buffer [20 mM Hepes (pH 7.2), 5 mM magnesium acetate, 125 mM potassium acetate, 0.4% Triton X-100, 1 mM DTT, supplemented with sodium orthovanadate (500 μM), phosphatase (Sigma), and protease (Roche) inhibitor cocktails], and equal aliquots of lysates were treated with 2 μg of anti-HA mAb (Covance) for 3 hours and protein G agarose beads for 45 min to immunoprecipitate p85α (PI3K). The bead-bound immune complexes were subsequently washed two times with each of the four different wash buffers in the following order: (i) PBS, 100 mM sodium orthovanadate, 1% Triton X-100; (ii) 100 mM tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium orthovanadate; (iii) TNE buffer [10 mM tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate]; and (iv) 20 mM Hepes (pH 7.5), 50 mM NaCl, 5 mM EDTA, 30 mM sodium pyrophosphate, 200 mM sodium orthovanadate, protease inhibitors, and 0.03% Triton X-100. The washed beads were resuspended in 70 μl of buffer and the reaction was started by the simultaneous addition of 10 μl of 10× ATP stock solution [65 mM Hepes (pH 7.0), 100 mM MgCl2, 500 μM ATP, and 10 μCi [γ-32P]ATP (specific activity >5000 Ci/mmol)] and 20 μl of freshly reconstituted l-α-phosphatidylinositol (1 mg/ml; from bovine liver, Sigma) in 20 mM Hepes (pH 7.0) and 1 mM EDTA. After incubation for 10 min at room temperature, the reactions were terminated by addition of 25 μl of 5 M HCl and vortexing. Lipids were extracted by addition of 160 μl of 1:1 mix of methanol/chloroform and vortexing. Organic and water-soluble phases were separated by centrifugation for 2 min at room temperature in a microfuge. Equal aliquots of the lower organic phase were loaded immediately onto thin-layer chromatography (TLC) plates (previously heat-activated at 100°C for 45 min) that were run in a tank equilibrated with a solvent mixture of chloroform/methanol/water/ammonium hydroxide (60:47:11.3:2) and subsequently analyzed by autoradiography.

Statistical analysis

Each experiment presented in the figures is representative of at least three independent experiments. Statistical significance (P value) between various conditions was assessed with one-way analysis of variance (ANOVA) and Bonferroni post hoc test. All graphical data presented were prepared with GraphPad Prism software (GraphPad Software Inc.).

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/4/192/ra64/DC1

Fig. S1. GIV is phosphorylated in vitro by receptor tyrosine kinases and in cells in response to growth factors.

Fig. S2. Bioinformatic analyses of the phosphopeptide sequences of GIV.

Fig. S3. Both EGFR and Src can phosphorylate GIV after EGF stimulation.

Fig. S4. The GEF domain is not required for tyrosine phosphorylation of GIV.

Fig. S5. GIV interacts with p85α and EGFR upon growth factor stimulation.

Fig. S6. Molecular modeling of the interfaces between the SH2 domains of p85α and the GIV-derived phosphopeptides.

Fig. S7. PIP3 production and localization as detected by a Btk-based reporter.

pTyrGIVCT files

References

References and Notes

  1. Acknowledgments: We thank M. G. Farquhar and G. N. Gill (UCSD) for scientific advice and thoughtful comments during the preparation of this manuscript; J. Wong and A. Ong (undergraduate students, UCSD) for assistance with protein expression and purification; and the members of the UCSD Mass Spectrometry Core Facility, of which M. Ghassemian is the director. Funding: P.G. was supported by the Burroughs Wellcome Fund, the Doris Duke Charitable Foundation, and a Research Scholar Award from the American Gastroenterology Association Foundation for Digestive Health and Nutrition. M.G.-M. was supported by Susan G. Komen Postdoctoral Fellowship KG080079. M.G. was supported by the Superfund Research Program. R.A. was supported by R01 GM 071872. Y.M. was supported by the Sarah Rogers Fellowship (UCSD). J.E. was supported by the McNair Scholarship Program. Author contributions: C.L., J.E., Y.P., Y.M., M.G.-M., and P.G. performed the experiments. I.K. and R.A. performed the computational modeling. M.G. performed the MS studies. Y.P., C.L., and J.E. prepared and characterized the protein constructs. C.L., J.E., Y.M., M.G.-M., and P.G. designed the experiments and analyzed the data. C.L., M.G.-M., and P.G. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data availability: The MS data associated with this manuscript may be downloaded from www.sciencesignaling.org/cgi/content/full/4/192/ra64/DC1.
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