Src Homology Domain 2-containing Protein-tyrosine Phosphatase-1 (SHP-1) Binds and Dephosphorylates Gα-interacting, Vesicle-associated Protein (GIV)/Girdin and Attenuates the GIV-Phosphatidylinositol 3-Kinase (PI3K)-Akt Signaling Pathway*

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.

Recent work has provided mechanistic insights into these biological functions of GIV. We previously demonstrated that GIV is a non-receptor guanine-nucleotide exchange factor (GEF) for G␣ i (7) and subsequently demonstrated that GIV directly binds ligand-activated epidermal growth factor receptor (EGFR) (2,13). By linking G protein signaling to EGFR and assembling a G␣ i -GIV-EGFR signaling complex, GIV enhances EGFR autophosphorylation, prolongs receptor association with the plasma membrane (PM) and selectively enhances PI3K-Akt signals initiated by ligand-activated receptors at the PM.
In depth understanding of how exactly GIV relays receptorinitiated signals selectively through the PI3K-Akt pathway remained poorly understood until recently when we defined (14) GIV as a central molecule in the tyrosine signaling pathway. We found that GIV is a common substrate for multiple receptor and non-receptor-tyrosine kinases and identified the sites of phosphorylation as tyrosines 1764 and 1798 in its C terminus. We demonstrated that these phosphotyrosines serve as docking sites for the two Src homology 2 (SH2) domains of the p85␣ regulatory subunit of PI3K, thereby allowing tyrosinephosphorylated GIV to directly bind and activate Class 1 PI3K downstream of both RTKs and GPCRs. We found that these molecular mechanisms orchestrate a distinct cascade of events when cells expressing wild-type GIV are stimulated with growth factors (2,14); GIV interacts with ligand-activated RTKs and enhances receptor autophosphorylation; subsequently, RTKs and non-RTKs phosphorylate GIV on two key tyrosines; phospho-GIV-PI3K complexes are assembled and stabilized at the receptor tail; receptor-initiated activation of PI3K is further enhanced by direct interaction of PI3K with phosphotyrosines within the C terminus of GIV, and enhanced PI3K activity generates PIP3 at the PM, which in turn triggers recruitment and activation of Akt. By contrast, in cells expressing a mutant GIV in which both tyrosines are replaced by phenylalanines, GIV interacts with ligand-activated RTKs, but the subsequent steps within the cascade are aborted. Furthermore, this "phospho-GIV-PI3K-Akt" cascade is progressively hyperactivated in breast cancer cells with advancing clinical stage and increasing degrees of tumor invasiveness (14), leading to the speculation that inhibition of tyrosine phosphorylation of GIV or disruption of the GIV-p85␣ interface are feasible therapeutic strategies to halt cancer progression. Despite the insights gained into which tyrosine kinases trigger GIV phosphorylation and its biological relevance during cancer invasion, the molecular mechanism(s) that down-regulates or attenuates this tyrosine-based signaling pathway remains unknown.
A major superfamily of proteins that down-regulate tyrosine-based signaling pathways by coordinately antagonizing the actions of protein-tyrosine kinases is protein-tyrosine phosphatases (PTPs) (15). Because of their antagonistic catalytic functions, PTPs and protein-tyrosine kinases act together to control a wide range of phosphotyrosine-mediated signaling pathways in mammalian cells. Of the PTPs known to date, we noted that the cytosolic PTP, Src homology 2 (SH2)-containing phosphatase-1 (SHP-1; also known as PTP1C, HCP, and SH-PTP1) stands out as the phosphatase that carries out a set of functions that antagonize almost all known functions of GIV during signal transduction. Most important of them all, whereas GIV has been shown to function as an enhancer of PI3K-Akt signals (2,5,7,14), SHP-1 is a major PTP that inhibits the PI3K-Akt pathway (16 -18). Although GIV binds the EGFR, enhances its autophosphorylation, and prolongs its signaling from the PM (2), SHP-1 binds to the activated receptor, dephosphorylates it, and promotes its endocytosis and degradation (19,20). Given these striking and diametrically opposite sets of functions, we wondered if SHP-1 may serve as the specific phosphatase that targets the phosphotyrosines in GIV and thereby down-regulates the phospho-GIV-PI3K-Akt axis.
In Vitro Phosphorylation and Dephosphorylation Assays-In vitro kinase-phosphatase assays were carried out in tandem using the following protocol. First, the kinase assays were performed using purified His-GIV-CT (ϳ3-5 g/reaction) and commercially obtained recombinant tyrosine kinases (EGFR, Millipore; c-Src, Cell Signaling). Reactions were started by the addition of 1 mM ATP and carried out at 25°C for 60 min in tyrosine kinase buffer (60 mM HEPES, pH 7.5, 5 mM MgCl 2 , 5 mM MnCl 2 ). Phosphorylated His-GIV-CT was subsequently used as the substrate in in vitro dephosphorylation assays using either purified His-SHP-1 (Fig. 1A), GST-SHP-1 WT, GST-SHP-1 C453S (Fig. 1C), HA-SHP-1 immuno-isolated from COS7 cells (Fig. 1D), or lysates of COS7 cells immuno-depleted of endogenous SHP-1 (Fig. 1F) as the source for phosphatase. Phosphatase reactions were carried out at 30°C for 60 min in phosphatase buffer (25 mM HEPES, 2.5 mM EDTA, 5 mM DTT, 50 mM NaCl, 65 ng/l BSA at pH 7.4) and stopped by adding Laemmli sample buffer and boiling at 100°C.
Cell Culture, Transfection, and Lysis-COS7 and HeLa cells were grown at 37°C in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, 1% L-glutamine, and 5% CO 2 . siRNA transfection of HeLa cells was carried out using Oligofectamine (Invitrogen) following the manufacturer's protocol. Oligos against human SHP-1 were from Santa Cruz Biotechnology. Briefly, HeLa cells at 70% confluence were transfected with 0.83 nM (final) of SHP-1 siRNA in Opti-MEM. Transfection of COS7 cells with HA-SHP-1, GIV-3ϫFLAG, or c-Src-HA were carried out using GeneJuice (Novagen). Lysates used as a source for SHP-1 for in vitro dephosphorylation assays or as a source for GIV in immunoprecipitation assays were prepared by resuspending cells in lysis buffer (20 mM HEPES, pH 7.2, 5 mM Mg(CH 3 COO) 2 , 125 mM K(CH 3 COO), 0.4% Triton X-100, 1 mM DTT supplemented with sodium orthovanadate (500 M), phosphatase (Sigma), and protease (Roche Diagnostics) inhibitor cocktails) and subsequently passing them through a 30-gauge needle at 4°C and cleared (centrifuged at 14,000 ϫ g for 10 min) before use in subsequent experiments. Whole cell lysates used to study the extent of Akt and ERK phosphorylation were prepared by resuspending the entire cell pellet in Laemmli sample buffer and boiling at 100°C.
In Vivo Phosphorylation Assays-For in vivo phosphorylation assays on endogenous GIV, HeLa cells were serum-starved for 12-16 h before stimulation with 50 nM EGF (Invitrogen). For in vivo phosphorylation assays using overexpressed GIV, FLAGtagged GIV was co-expressed with either EGFR, Src, or SHP-1 plasmids in COS7 cells in various assays. ϳ30 h after transfection, cells were serum-starved (0% FBS) for an additional 16 -18 h followed by stimulation with 50 nM EGF or 10 M LPA. Reactions were stopped using PBS chilled at 4°C supplemented with 200 M sodium orthovanadate and immediately scraped and lysed for immunoprecipitation.
Immunoprecipitation and GST Pulldown Assays-These assays were carried out exactly as described previously (2,9). Briefly, cell lysates (ϳ1-2 mg of protein) were incubated for 4 h at 4°C with 2 g of anti-HA mAb (Covance), anti-FLAG mAb (Sigma), 1.6 g of anti-SHP-1 PTP pAb, or anti SHP-2 PTP pAb, or 1 g of their respective preimmune IgGs followed by incubation with protein G (for all mAbs) or A (for all pAbs)-Sepharose beads (GE Healthcare) at 4°C for an additional 60 min. Beads were washed in PBS-T wash buffer (4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% (v:v) Tween 20, 10 mM MgCl 2 , 5 mM EDTA, 2 mM DTT, and 0.5 mM sodium orthovanadate), and bound proteins were eluted by boiling in Laemmli sample buffer. In vitro binding assays with GST-fused proteins were carried out exactly as previously described (7,9). Buffers were supplemented with 0.5 mM sodium orthovanadate for all steps of the assay.
Statistical Analysis-Each experiment presented in the figures is representative of at least three independent experiments. Statistical significance between various conditions was assessed with the Wilcoxon Signed-Rank test. Graphical data presented was prepared using GraphPad Software, Inc. (San Diego, CA).

RESULTS
Protein-tyrosine Phosphatase SHP-1 Dephosphorylates GIV in Vitro-To determine whether GIV is a substrate of SHP-1, we carried out in vitro phosphatase assays using bacterially expressed, recombinant His-SHP-1 and tyrosine-phosphorylated His-GIV-CT (amino acids 1660 -1870) as substrate. We examined the C terminus of GIV because the only two sites of tyrosine phosphorylation in the entire protein are located within that region (14). To generate the phosphorylated GIV-CT substrate, we first carried out in vitro kinase assays using His-GIV-CT and recombinant EGFR kinase (Millipore) as described previously (14). When phosphorylated, His-GIV-CT was subsequently incubated with increasing quantities of His-SHP-1, SHP-1 efficiently dephosphorylated GIV in a dose-dependent manner (Fig. 1A) indicating that GIV is a substrate of SHP-1 in vitro.
Because GIV is tyrosine-phosphorylated at two sites, Tyr-1764 and -1798 (14), next we asked if SHP-1 selectively dephosphorylates one or both of those phosphotyrosines. When phospho-mutants of His-GIV-CT with either Tyr-1764 or -1798 mutated to phenylalanine were phosphorylated in vitro by EGFR kinase and subsequently incubated with His-SHP-1, SHP-1 efficiently dephosphorylated both His-GIV-CT mutants
To investigate if the catalytic activity of SHP-1 is required to dephosphorylate GIV, in vitro phosphatase assays were carried out using phosphorylated His-GIV-CT and GST-tagged SHP-1 WT or the constitutively inactive mutant in which cysteine 453 in the catalytic center is replaced with serine (SHP-1 C453S, hereby referred to as CS). The CS mutation selectively abolishes PTP activity while retaining the ability of SHP-1 to bind substrate proteins (24). SHP-1 WT, but not SHP-1 CS dephosphorylated tyrosine-phosphorylated His-GIV-CT in a dose-dependent manner (Fig. 1C). Identical results were obtained when SHP-1-HA immuno-isolated from COS7 cells was used to dephosphorylate EGFR-or Src-phosphorylated His-GIV-CT in vitro (Fig. 1D). These results demonstrate that an intact catalytic domain is indeed required for the protein-tyrosine phosphatase SHP-1 to dephosphorylate GIV-CT in vitro.
Next we asked whether other tyrosine phosphatases besides SHP-1 can dephosphorylate GIV. To this end we prepared mock-depleted and SHP-1-depleted COS7 lysates (Fig. 1E) and used them as sources of phosphatases in in vitro phosphatase assays with tyrosine-phosphorylated His-GIV-CT. Although mock-depleted lysates efficiently dephosphorylated tyrosinephosphorylated GIV-CT, lysates immuno-depleted of SHP-1 (by Ͼ99%) failed to do so (Fig. 1F). Furthermore, the addition of recombinant His-SHP-1 to SHP-1-depleted lysates restored the ability of this lysate to dephosphorylate tyrosine-phosphorylated His-GIV-CT (Fig. 1F). Thus, the key catalytic activity that is required for removal of the phosphotyrosines in GIV was lost with depletion of SHP-1 and restored by adding back recombinant His-SHP-1. These results indicate that SHP-1 is the major cellular phosphatase that dephosphorylates the tyrosines in GIV in vitro. Of note, SHP-2, the closest relative of SHP-1, was abundant in both mock-and SHP-1-depleted cytosols (Fig. 1E) but failed to dephosphorylate His-GIV-CT (Fig. 1F), highlighting the specificity of SHP-1 toward GIV-CT as its substrate.
SHP-1 Inhibits Tyrosine Phosphorylation of GIV after Activation of EGF Receptor or Src Kinases-We recently demonstrated that both receptor (EGFR and InsulinR) and non-receptor (c-Src) tyrosine kinases can phosphorylate the GIV C-terminal tyrosines in vivo (14). To determine whether SHP-1 can inhibit EGFR-or Src-induced tyrosine phosphorylation of GIV in cells, we carried out in vivo phosphorylation assays using an approach identical to what we used previously (14). When we immunoprecipitated GIV from COS7 cells co-transfected with GIV-FLAG and HA-SHP-1 and assessed the extent of tyrosine phosphorylation on GIV, we found that the co-expression of SHP-1 virtually abolished the EGF-triggered tyrosine phosphorylation (supplemental Fig. S1). When we performed the in vivo phosphorylation assay in cells co-transfected with either HA-SHP-1-WT or HA-SHP-1-CS mutant, we found that WT, but not the CS, mutant virtually abolished the EGF-triggered tyrosine-phosphorylation ( Fig. 2A), thus confirming that the catalytic activity of SHP-1 is required for down-regulation of tyrosine-phosphorylated GIV after EGF stimulation. We conclude that SHP-1 dephosphorylates tyrosine-phosphorylated GIV downstream of EGFR.
To discern if SHP-1 can also dephosphorylate GIV after activation of non-RTKs like Src, we immunoprecipitated GIV from COS7 cells co-expressing GIV-FLAG with constitutively active (Y527F (25)) or inactive (K295R (26)) mutants of Src-HA and the WT or the inactive CS mutant of HA-SHP-1. As shown previously by us (14), GIV was tyrosine-phosphorylated in a Src activity-dependent manner; co-transfection of GIV with active, but not inactive Src mutant resulted in increased tyrosine phosphorylation of GIV (Fig. 2B). Co-expression of SHP-1 WT, but not SHP-1 CS abolished Src-dependent tyrosine phosphorylation of GIV (Fig. 2B), demonstrating that the catalytic activity of SHP-1 is required for down-regulation of Srcphosphorylated GIV. We conclude that SHP-1 antagonizes the action of Src kinase by dephosphorylating GIV once it is phosphorylated by Src.
To investigate how SHP-1 affects the timing and extent of tyrosine phosphorylation of endogenous GIV, we depleted HeLa cells of endogenous SHP-1 using target-specific or control siRNA. We subsequently immunoprecipitated GIV and assessed the extent of tyrosine phosphorylation at 5 or 15 min after EGF stimulation. Consistent with our recent work (14), in control siRNA-treated cells tyrosine phosphorylation of GIV occurred exclusively after ligand stimulation, reached peak levels at 5 min, and was down-regulated at 15 min (Fig. 2C). In cells depleted of SHP-1 (by ϳ70%), although the timing of phosphodephosphorylation of GIV remained same, the extent of peak phosphorylation was enhanced by ϳ4.2-fold at 5 min and ϳ1.5fold at 15 min compared with controls. Of note, levels of total EGFR, the peak extent of receptor autophosphorylation (Tyr(P) EGFR), and the rate of receptor degradation were almost simi-lar in both control and SHP-1-depleted cells at 5 min and only slightly increased in SHP-1-depleted cells at 15 min, indicating that the tyrosine kinase activity of EGFR was similar under these conditions. This indicates that the observed increase in the abundance of tyrosine-phosphorylated GIV in cells depleted of SHP-1 at 5 min is unlikely to be due to hyperphosphorylation by EGFR and instead is a consequence of impaired dephosphorylation of GIV by SHP-1. Taken together, these results demonstrate that SHP-1 can dephosphorylate GIV in vivo after activation of both RTKs and non-RTKs.
SHP-1 Inhibits Tyrosine Phosphorylation of GIV Downstream of G Protein-coupled Receptors-Because activation of GPCRs is known to trigger tyrosine phosphorylation of GIV (via Src kinase) (14) and to activate SHP-1 (via G␣q) (27), next we investigated if SHP-1 can down-regulate tyrosine phosphorylation of GIV after activation of lysophosphatidic acid (LPA) receptor, a member of the GPCR family. Consistent with our recent work (14), activation of LPA receptor triggered tyrosine phosphorylation of GIV (supplemental Fig.  S2, Fig. 3). This LPA-triggered tyrosine phosphorylation was inhibited in cells co-expressing SHP-1 WT but maintained in those cells co-expressing the inactive SHP-1 CS mutant (Fig. 3), indicating that the catalytic activity of SHP-1 is required for down-regulation of tyrosine-phosphorylated GIV after LPA stimulation. We conclude that SHP-1 also dephosphorylates GIV downstream of GPCRs.

SHP-1 Interacts Directly and Constitutively with GIV-Most
PTPs interact with their substrates before catalyzing the removal of phosphates on target tyrosines (28,29). To investigate if endogenous SHP-1 and GIV interact in vivo, we immunoprecipitated SHP-1 from COS7 (Fig. 4A) or HEK (supplemental Fig. S3) cells and immunoblotted for GIV. GIV coimmunoprecipitated with SHP-1 in both cell lines, indicating that they interact at steady state in vivo. Of note, this GIV-SHP-1 interaction is specific because GIV did not co-immunoprecipitate with the closely related family member SHP-2 (Fig.  4A). To assess if the GIV-SHP-1 interaction is ligand-dependent, we immunoprecipitated SHP-1 from serum-starved or EGF-stimulated COS7 cells and analyzed the immune complexes for GIV. We found that the ratio of GIV:SHP-1 in immune complexes remained unchanged before and after EGF stimulation, indicating that the interaction occurs constitutively, i.e. independent of receptor activation. The abundance of GIV-SHP-1 interaction also remained unchanged regardless of the activation status of Src (data not shown). In contrast to GIV, G␣ i3 did not interact with SHP-1 in either COS7 or HEK cell lines (Fig. 4B, supplemental Fig. S3). To determine whether the GEF motif of GIV, via which GIV activates G␣ i subunits (1-3, 7), is required for the GIV-SHP-1 interaction, we immunoprecipitated SHP-1 from COS7 cells expressing WT or the GEFdeficient F1685A (FA) mutant of GIV-FLAG and analyzed the immune complexes for GIV-FLAG. We found that the relative abundance of GIV:SHP-1 complexes was similar between GIV-FA and GIV-WT (Fig. 4C), demonstrating that GIV GEF motif and GIV-dependent activation of G␣ i are not required for GIV to interact with SHP-1 in cells. Taken together, we conclude that GIV, but not G␣ i3 , interacts specifically with SHP-1 (and not SHP-2) in mammalian cells, and this interaction occurs constitutively, i.e. independent of both receptor and G protein activation.
To determine whether the GIV-SHP-1 interaction we observe in vivo is direct, we carried out in vitro binding assays using recombinant His-GIV-CT and GST or GST-SHP-1 immobilized on glutathione beads. GST-SHP-1, but not GST alone, bound His-GIV-CT (Fig. 5A), indicating that SHP-1 directly binds GIV and that the C terminus of GIV is sufficient to mediate such an interaction. Because SHP-1 is recruited to its substrates by one or both of its in-tandem SH2 domains (30), next we asked if the SH2 domains (if so, which one (NSH2 or CSH2)) is responsible for mediating the direct interaction of SHP-1 with the C terminus of GIV. For this we generated GSTtagged truncated versions of SHP-1 lacking either both SH2  4 and 6). Activation of EGFR and depletion of SHP-1 (by ϳ70%) were confirmed by analyzing equal aliquots of lysates for GIV, total EGFR (t EGFR), phosphotyrosine 1173 EGFR (pY EGFR), SHP-1, G␣ i3 , and tubulin by immunoblotting.
domains (⌬NϩC SH2) or just the NSH2 domain (⌬NSH2, in which the CSH2 is intact) and used them alongside GST-SHP-1 (full-length) in binding assays. When these GST-SHP-1 proteins were immobilized on glutathione beads and incubated with His-GIV-CT, GIV-CT bound equally to full-length SHP-1 and to SHP-1 ⌬NSH2, whereas binding to SHP-1 ⌬NϩCSH2 was dramatically reduced by ϳ80% (Fig. 5B), indicating that the NSH2 domain is not required for SHP-1 to bind GIV, but the CSH2 domain is, and that the CSH2 domain can account for the observed interaction between GIV-CT and SHP-1 in vitro. Thus, the second (CSH2) but not the first (NSH2) of the two in-tandem SH2 domains of SHP-1 directly binds the C terminus of GIV. Noteworthy, the observed interaction between GST-SHP-1 and His-GIV-CT was not affected by tyrosine phosphorylation of GIV-CT (data not shown). Taken together with our findings by co-immunoprecipitation assays on cell lysates (Fig. 4), we conclude that the GIV-SHP-1 interaction is direct and constitutive. (14) that tyrosine-phosphorylated GIV directly binds the p85␣-regulatory subunit of PI3K, activates Class 1 PI3K, enhances the generation of second messenger PIP3 at the PM, and triggers the subsequent recruitment and activation of Akt kinase. Because SHP-1 dephosphorylates tyrosine-phosphorylated GIV, we predicted that SHP-1 might also inhibit the ligand-dependent assembly of GIV-PI3K complexes in cells. We found that is indeed the case because EGF-induced formation of complexes between p85␣(PI3K) and endogenous GIV (Fig. 6) or FLAG-tagged GIV (supplemental Fig. S4) in COS7 cells was inhibited in the presence of WT but not the inactive CS mutant of HA-SHP-1. Of note, the failure to assemble GIV-p85␣(PI3K) complexes in cells expressing SHP-1 WT coincided with a dramatic suppression of tyrosinephosphorylated GIV to virtually undetectable levels. These findings indicate that the catalytic activity is required for SHP-1 to simultaneously dephosphorylate tyrosine-phosphorylated GIV and to inhibit the formation of phospho-GIV-p85␣ complexes. These results indicate that the catalytic activity of SHP-1 is required to dephosphorylate the phosphotyrosines in GIV and thereby inhibit the formation of phospho-GIV-p85␣ complexes.

SHP-1 Attenuates GIV-dependent Enhancement of PI3K-Akt Signals by Dephosphorylating Tyrosine-phosphorylated GIV and Inhibiting the Assembly of Phospho-GIV-p85␣(PI3K) Complexes-We recently demonstrated
To determine whether SHP-1 affects GIV function as an enhancer of Akt signaling, we co-transfected COS7 cells with GIV-FLAG and either SHP-1-WT or SHP-1-CS mutant and maintained the cells in 2% FBS before lysis. Consistent with the previously established role of GIV as an enhancer of Akt phosphorylation (5,7,14), we found that overexpression of GIV-FLAG alone enhanced Akt activity by ϳ2.5-fold, as determined by the extent of Akt phosphorylation at Ser-473 (Fig. 7, A and  B). Co-expression of WT, but not the inactive CS mutant of SHP-1, returned Akt activity to levels seen in control cells without GIV-FLAG (Fig. 7, A and B), indicating that a catalytically active SHP-1 is required to inhibit GIV from enhancing the phosphorylation and subsequent activation of Akt kinase. Furthermore, depletion of endogenous SHP-1 from HeLa cells by siRNA was associated with enhanced Akt phosphorylation at both 5 and 15 min after EGF stimulation (Fig. 7C), temporally coinciding with the enhanced phosphorylation of GIV observed in these cells at those time points (Fig. 2C). Of note, the peak phosphorylation of ERK1/2 in SHP-1-depleted cells was similar to controls, indicating that the observed effect of SHP-1 on Akt signaling is pathway-specific. Based on these findings, we conclude that SHP-1 antagonizes/attenuates GIV-dependent enhancement of Akt signaling and that enhancement of Akt activity in the absence of SHP-1 is mediated at least in part due to a failure to down-regulate tyrosine-phosphorylated GIV and the phospho-GIV-PI3K-AKt signaling cascade.
In summary (Fig. 8), here we demonstrate that SHP-1 is the major cellular phosphatase that down-regulates tyrosine-phosphorylated GIV downstream of both growth factor RTKs and GPCRs and attenuates the previously characterized (14) prometastatic phospho-GIV-PI3K-Akt axis of signaling.

DISCUSSION
The key finding in this work is the identification of SHP-1 as the major and specific PTP that dephosphorylates GIV and attenuates GIV-dependent enhancement of the PI3K-Akt signaling pathway. We previously demonstrated (14) that ligand stimulation of either RTKs or GPCRs leads to tyrosine phosphorylation of GIV by cooperative action of activated RTKs and non-RTKs, triggers the formation of phospho-GIV-PI3K complexes via two key phosphotyrosines within the C terminus of GIV that serve as docking sites for p85␣(PI3K), and thereby enhances the activity of PI3K and Akt kinases. Here we show that ligand stimulation of either RTKs or GPCRs also simultaneously activates SHP-1, which binds and dephosphorylates tyrosine-phosphorylated GIV, dissociates GIV-PI3K complexes, and prevents GIV from enhancing the activity of PI3K, thereby serving as a negative feedback for down-regulating the receptor-GIV-PI3K axis of signal transduction. Our results also demonstrate that SHP-1 is the specific PTP that dephosphorylates both tyrosines in the GIV C terminus and that SHP-1 dephosphorylates these tyrosines irrespective of whether they are phosphorylated by receptor (EGFR) or non-receptor (Src) tyrosine kinase. We conclude that regardless of the type of receptor activated, SHP-1 attenuates the GIV-PI3K-Akt axis of signaling by antagonizing the action of protein-tyrosine kinases on GIV.
We also demonstrate that SHP-1 directly and constitutively binds the C terminus of GIV. By domain mapping, we further established that this interaction is mediated maximally via the second of the two in-tandem SH2 domains (CSH2) in SHP-1. Although SH2 domains commonly recognize and bind phosphorylated tyrosines, thereby triggering the formation of protein complexes in a ligand-dependent manner (31), in the case of the SHP-1 SH2 domains, a preference for phosphorylated GIV in vitro or ligand dependence in cells was not observed. However, this is not unusual because the SH2 domains of SHP-1 are known to often bind interacting partners/substrates in a phosphotyrosine-independent manner (32-37), including its own C terminus (38). Our immunoprecipitation assays demonstrate that only a portion of GIV and SHP-1 molecules is constitutively associated (Fig. 4). Given that both the C terminus of GIV (2) and the SH2 domains of SHP-1 (19,20) can independently and directly bind EGFR via phosphotyrosines in the cytoplasmic tails of the ligand-activated receptor, our findings are compatible with the possibility that in unstimulated cells the SHP-1-GIV interaction is direct and is independent of receptor, whereas ligand stimulation may trigger the formation of ternary complexes between autophosphorylated EGFR, the C terminus of GIV, and SHP-1 via three-way direct or indirect interactions; (i) GIV and Tyr(P)-1148 EGFR (2), 5 (ii) SHP-1(NSH2) and Tyr(P)-1173 EGFR (19,20), and (iii) GIV and SHP-1(CSH2) (this work). Although these receptor-dependent interactions are acute and are mostly implicated in fine-tuning receptor autophosphorylation and down-regulation of receptor signaling (2,19,20), the receptor-independent, direct and constitutive association between GIV and SHP-1 we report here may allow a more flexible and diverse regulation of both GIV functions and SHP-1 activity.
With regard to the biological significance of such partial constitutive interaction, there are several possibilities. We identify one major consequence of such an interaction as dephosphorylation and down-regulation of tyrosine-phosphorylated GIV by SHP-1. Besides ensuring a rapid down-regulation of tyrosinebased signaling by GIV, another possible consequence of the constitutive proximity of SHP-1 to its substrates could be that such a setup allows the substrate to feedback regulate SHP-1 catalytic activity, as seen in other instances (32,35,37,39). For example, GIV interaction with the CSH2 domain of SHP-1 could "release" the conformation-dependent autoinhibition of SHP-1 by its in-tandem NSH2 domain (38), as shown for RTKs (38). Noteworthy, we did not find any homology between GIV sequences flanking its two C-terminal phosphotyrosines and the conserved immunoreceptor tyrosinebased inhibition motifs that are found in the cytoplasmic tails of many receptors of the immune system that bind to the CSH2 domain of SHP-1 and activate the phosphatase (40). Thus, it is unlikely that binding of GIV to the CSH2 domain directly activates the catalytic activity of SHP-1. However, it is tempting to speculate that the ability of GIV to bind the CSH2 domain of SHP-1 might affect the ability of other immunoreceptor tyrosine-based inhibition motifs to activate the phosphatase in cells, and this might be another mechanism via which GIV may interfere with activation of SHP-1 after receptor stimulation and further enhance phosphotyrosine-based signaling.
We also define the molecular mechanism by which SHP-1 antagonizes the phospho-GIV-PI3K-Akt axis of signaling. Here we demonstrate that by dephosphorylating phosphotyrosines   2 and 3), undetectable in cells expressing SHP-1 WT but restored in cells expressing SHP-1 C453S. Ab, antibody. phosphotyrosines of GIV that serve as docking sites for the SH2 domains of p85␣-regulatory subunit of Class 1A PI3Ks, SHP-1 inhibits the formation of GIV-PI3K complexes and abrogates phospho-GIV-dependent enhancement of PI3K activity and inhibits the subsequent phosphorylation and activation of Akt in cells. We also demonstrate that SHP-1, but not its closest structurally related family member, SHP-2, is the specific phosphatase for GIV. This is in keeping with the fact that whereas SHP-1 antagonizes Akt signaling (18,(41)(42)(43)(44) and inhibits chemotaxis (27,(45)(46)(47)(48), SHP-2 is frequently associated with enhancement of the PI3K-Akt pathway (49 -53) during growth factor-initiated cell migration (54). Of note, all of the experimental evidence supporting SHP-1 ability to antagonize the Akt pathway was obtained using mesenchymal and hematopoietic cells and the biological role of the epithelium-specific isoform of SHP-1 and how it might inhibit chemotaxis/cell migration remained elusive (55). We show here that in epithelial cells depleted of SHP-1, both peak tyrosine phosphorylation of GIV and Akt activity are coincidently enhanced. By contrast, in cells expressing a constitutively dephosphorylated mutant GIV in which both tyrosines 1764 and 1798 are mutated to phenylalanines, PI3K-Akt signals are abrogated, actin remodeling fails to generate stress fibers, and consequently, cells do not migrate efficiently in scratch-wound assays (14). Noteworthy, these phenotypes (Akt enhancement, actin remodeling, and cell migration) have been previously implicated in contributing to the prometastatic functions of GIV expressed in tumor cells (1-4, 7, 11, 12). These findings together with the data presented in this work define the biological importance of the epithelial isoform of SHP-1 (55).
Our findings also underscore the clinical significance of SHP-1 in antagonizing the previously defined prometastatic functions of GIV that are expressed in the tumor epithelium (1)(2)(3)(4)12). GIV and SHP-1 display a striking contrast with regard to their level of expression and role in tumor cell invasion and angiogenesis during cancer progression (expression of GIV mRNA and protein increase during cancer progression (2,3,12)), and GIV is essential for tumor cell invasion and neoangiogenesis (3,4), whereas expression of SHP-1 is suppressed during cancer progression, and SHP-1 is known to inhibit angiogenesis (57)(58)(59)(60)(61)(62). Because the role of GIV-dependent PI3K-Akt enhancement and cell migration extends beyond tumor cell invasion/metastasis into other diverse biological processes, e.g. epithelial wound healing (1,8), neuronal migration (10,63,64), cellular autophagy (6), maintenance of vascular integrity after injury (65), and neoangiogenesis (4), it is plausible that SHP-1 antagonizes GIV functions during all these processes via a common underlying mechanism outlined here.
In conclusion, we not only identify SHP-1 as the phosphatase that dephosphorylates GIV and elucidate the molecular mechanism(s) by which it attenuates a major tyrosine-based signaling pathway, but we also provide insights into the hitherto elusive role of epithelial SHP-1.