Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway

Abstract

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.

FIGURE 1.

SHP-1 specifically dephosphorylates tyrosine-phosphorylated GIV in vitro. A, His-SHP-1 dephosphorylated tyrosine-phosphorylated His-GIV-CT. His-GIV-CT (1660–1870) was phosphorylated in vitro using recombinant EGFR kinase and was subsequently used as the substrate in phosphatase assays using increasing (1, 2, and 4 μg) amounts of recombinant His-SHP-1. Residual tyrosine phosphorylation on His-GIV-CT was assessed by immunoblotting (IB) using phosphotyrosine (pTyr) and His mAbs. His-GIV-CT was efficiently dephosphorylated in the presence of 1 μg and maximally in the presence of 2 μg of His-SHP-1. B, SHP-1 dephosphorylated both phosphotyrosines (Tyr-1764 and -1798) in the C terminus of GIV. His-GIV-CT mutants with one intact tyrosine (Y1764F mutant, in which Tyr-1798 is intact, and Y1798F mutant, in which Tyr-1764 is intact) were phosphorylated in vitro using recombinant EGFR kinase as in A. Equal aliquots (5 μg) of these phosphorylated His-GIV-CT mutants were subsequently used in phosphatase assays in the presence (+) or absence (−) of 1 μg His-SHP-1 and analyzed for residual tyrosine phosphorylation by immunoblotting as in A. C, WT, but not the catalytically inactive CS mutant of SHP-1 dephosphorylates tyrosine-phosphorylated His-GIV-CT in vitro. Equal aliquots of tyrosine-phosphorylated His-GIV-CT (phosphorylated using recombinant EGFR kinase as in A) were used as substrates in phosphatase assays with the indicated amounts of bacterially expressed, purified WT, or C453S mutant of GST-SHP-1. Samples were subsequently analyzed for residual tyrosine phosphorylation by immunoblotting as in A. GST-SHP-1 WT efficiently dephosphorylated His-GIV-CT at 1 μg (lane 3) and maximally at 3 μg (lane 4), whereas GST-SHP-1 CS failed to dephosphorylate His-GIV-CT at either dose (lanes 5 and 6). D, WT, but not the catalytically inactive CS mutant of SHP-1, immuno-isolated from COS7 cells dephosphorylated tyrosine-phosphorylated His-GIV-CT in vitro. Tyrosine-phosphorylated His-GIV-CT was generated by carrying out in vitro phosphorylation assays using recombinant EGFR (pY-GIV-CT (EGFR); lanes 1–3) or Src (pY-GIV-CT (Src); lanes 4–6) kinases as in A. Lysates of COS7 cells transiently expressing WT or the CS mutant of HA-SHP-1 were incubated sequentially with anti-HA mAb and protein G-agarose beads to immuno-isolate active (WT) and catalytically inactive CS phosphatase, respectively. In vitro phosphatase assays were subsequently carried out by incubating equal aliquots of Tyr(P)-GIV-CT with the bead-bound SHP-1 WT (lanes 2 and 4), SHP-1 CS (lanes 3 and 5), or control beads (lane 1). Both EGFR and Src-phosphorylated GIV-CT were efficiently dephosphorylated in the presence of HA-SHP-1 WT (lanes 2 and 4) but not HA-SHP-1 CS (lanes 3 and 5) or control beads (lane 1). E and F, COS7 cell lysates immuno-depleted of endogenous SHP-1 fail to dephosphorylate tyrosine-phosphorylated His-GIV-CT in vitro. E, lysates of COS7 cells were immuno-depleted of SHP-1 (lane 2) or mock-depleted (lane 1) using anti-SHP-1 or preimmune control IgGs, respectively. Equal aliquots of lysates were analyzed for GIV, SHP-1, SHP-2, and actin by immunoblotting. The efficacy of SHP-1 immuno-depletion was confirmed as ∼>99% by optical densitometry. F, equal aliquots of tyrosine-phosphorylated His-GIV-CT (∼5 μg; prepared as in A) were used as substrates in in vitro dephosphorylation assays with either buffer alone (lane 5) or equal aliquots (∼75 μg) of mock-depleted (lanes 1 and 2) or SHP-1-depleted (lanes 3 and 4) lysates. Residual phosphorylation was assessed by immunoblotting for Tyr(P) mAb. Tyrosine-phosphorylated His-GIV-CT was efficiently dephosphorylated in the presence of mock-depleted (lane 2) but not SHP-1-depleted lysate (lane 3). The phosphatase activity of SHP-1-depleted lysate was restored upon the addition of bacterially expressed His-SHP-1 (lane 4).

FIGURE 2.

SHP-1 inhibits tyrosine phosphorylation of GIV by EGFR and Src. A, WT, but not the catalytically inactive CS mutant of SHP-1, dephosphorylates GIV after EGF stimulation. COS7 cells transfected with vector alone (lane 1), GIV-FLAG alone (lanes 2 and 3), GIV-FLAG and HA-SHP-1 WT (lane 4), or GIV-FLAG and HA-SHP-1 CS mutant (lane 5) were serum-starved (−) and subsequently stimulated with EGF (50 nm, +) for 10 min. Equal aliquots of lysates (bottom) were incubated sequentially with anti-FLAG mAb and protein G-agarose beads. Immune complexes (top) were analyzed by immunoblotting (IB) for GIV and Tyr(P) (pTyr) using the Li-COR Odyssey Infrared Western blot Imaging System. Single channel images for GIV and Tyr(P) are displayed in grayscale, which shows that immunoprecipitated GIV is phosphorylated on tyrosine(s) exclusively after EGFR stimulation (compare lanes 2 and 3). This EGF-dependent tyrosine phosphorylation of GIV was undetectable in cells co-transfected with HA-SHP-1 WT (lane 4) but robust in those expressing HA-SHP-1 CS (lane 5). Expression of GIV and SHP-1 in all lysates was analyzed by immunoblotting for FLAG, GIV, SHP-1, and tubulin (bottom). B, WT, but not the catalytically inactive CS mutant of SHP-1, dephosphorylates Src-phosphorylated GIV. COS7 cells were transfected with vector alone (lane 1) or GIV-FLAG alone (lane 2) or GIV-FLAG and Src-HA K295R inactive mutant (In, lane 3), or GIV-FLAG and Src-HA Y527F active mutant (Ac, lanes 4–6). HA-SHP-1 WT and HA-SHP-1 CS were co-transfected with active Src Y527F in lanes 5 and 6, respectively. Equal aliquots of lysates (bottom) were incubated with anti-FLAG mAb and protein G-agarose beads. Immune complexes (top) were analyzed by immunoblotting for GIV and Tyr(P) as in A. Tyrosine phosphorylation was detectable in GIV-FLAG immunoprecipitated from cells co-expressing the active Src-HA mutant (lane 4, pTyr panel) but not from cells expressing vector (lane 1) or GIV-FLAG alone (lane 2) or those expressing the inactive Src-HA mutant (lane 3). Src-induced tyrosine phosphorylation of GIV-FLAG was undetectable in cells co-expressing HA-SHP-1 WT (lane 5) but restored in cells co-expressing HA-SHP-1 CS (lane 6). Expression of GIV, Src, and SHP-1 in all lysates was analyzed by immunoblotting for FLAG, GIV, SHP-1, HA (Src), and tubulin (bottom). C, EGF-dependent tyrosine-phosphorylation of GIV is enhanced in cells depleted of endogenous SHP-1. HeLa cells treated with scrambled (Scr) or SHP-1 siRNA were serum-starved (0 min) followed by stimulation with 50 nm EGF for 5 and 15 min. Equal aliquots of lysates (left) were incubated sequentially with anti-GIV-CT and protein A-agarose beads. Immune complexes (right) were analyzed for GIV and Tyr(P) by immunoblotting. Tyrosine-phosphorylated GIV (pTyr) was undetectable in starved cells (lane 2), peaked at 5 min after EGF stimulation in both scrambled and SHP-1 siRNA-treated cells (lanes 3 and 5), and decreased at 15 min (lanes 4 and 6). Phosphorylation of GIV in the immunoprecipitates (right panel) is increased in SHP-1-depleted cells as compared with control by ∼4.2-fold at 5 min (compare lanes 3 and 5) and by ∼1.5-fold at 15 min (compare lanes 4 and 6). Activation of EGFR and depletion of SHP-1 (by ∼70%) were confirmed by analyzing equal aliquots of lysates for GIV, total EGFR (t EGFR), phosphotyrosine 1173 EGFR (pY EGFR), SHP-1, Gα_i3, and tubulin by immunoblotting.

FIGURE 3.

WT, but not the catalytically inactive SHP-1C453S (CS) mutant, inhibits tyrosine phosphorylation of GIV upon activation of LPA receptor. COS7 cells transfected with vector alone (lane 1), GIV-FLAG alone (lanes 2 and 3), GIV-FLAG and HA-SHP-1 WT (lane 4), or GIV-FLAG and HA-SHP-1 CS mutant (lane 5) were serum-starved (−) and subsequently stimulated with LPA (10 μm, +) for 20 min. Equal aliquots of lysates (bottom) were incubated with anti-FLAG mAb and protein G-agarose beads. Immune complexes (top) were analyzed for GIV and Tyr(P) (pTyr) by immunoblotting. Immunoprecipitated GIV was phosphorylated on tyrosine(s) exclusively after LPA receptor stimulation (compare lanes 2 and 3). LPA-dependent tyrosine phosphorylation of GIV was markedly reduced in cells co-transfected with HA-SHP-1 WT (lane 4) but robust in those expressing HA-SHP-1 CS (lane 5). Adequate stimulation of cells by LPA and expression of GIV, Src, and SHP-1 in all lysates was analyzed by immunoblotting for FLAG, GIV, SHP-1, HA (Src), phospho-ERK1/2, and tubulin (bottom).

FIGURE 4.

SHP-1 interacts with GIV in vivo. A, GIV co-immunoprecipitates with SHP-1, but not SHP-2. Equal aliquots of COS7 lysates (left) were incubated with either rabbit preimmune (first lane) or anti-SHP-1 (second lane) or anti-SHP-2 (third lane) IgGs and protein A-agarose beads. Immune complexes (25) were analyzed for GIV, SHP-1, and SHP-2 by immunoblotting (IB). SHP-1 and SHP-2 were immunoprecipitated efficiently and specifically. GIV co-immunoprecipitated with SHP-1 (second lane) but not SHP-2 (third lane) or control IgG (first lane). B, GIV, but not Gα_i3, interacts constitutively with SHP-1. COS7 cells were serum-starved (−) and subsequently stimulated with 50 nm EGF for 10 min (+). Equal aliquots of lysates (bottom) were incubated with either rabbit preimmune (lane 1) or anti-SHP-1 (lanes 2 and 3) IgGs and protein A-agarose beads. Immune complexes (top) were analyzed for GIV, SHP-1, and Gα_i3 by immunoblotting. SHP-1 was immunoprecipitated efficiently and specifically. GIV, but not Gα_i3, co-immunoprecipitated with SHP-1 from both starved and EGF-stimulated cells (lanes 2 and 3). C, GIV GEF motif is not required for the GIV-SHP-1 interaction. COS7 cells were transfected with vector alone (lane 1) or FLAG-tagged wild-type GIV (GIV-WT; lane 2), or the GEF-deficient F1685A (FA) mutant (GIV-FA; lane 3). Equal aliquots of lysates (bottom) were incubated with anti-SHP-1 IgGs and protein A-agarose beads. Immune complexes were analyzed for FLAG (GIV-FLAG) and SHP-1 by immunoblotting.

FIGURE 5.

The C terminus of GIV directly binds to SHP-1. A, His-GIV-CT (amino acids 1660–1870) directly binds GST-SHP-1. Equal aliquots (∼3 μg) of His-GIV-CT were incubated with ∼30 μg of GST and ∼25 μg of GST-SHP-1 immobilized on glutathione beads. Bound His-GIV-CT was analyzed by immunoblotting (IB) with GIV-CT pAb. Relative amounts of bead-bound GST and GST-SHP-1 were confirmed by Ponceau S staining. B, His-GIV-CT preferentially binds the C-terminal SH2 domain of SHP-1. Equal aliquots (∼3 μg) of His-GIV-CT were incubated with ∼30 μg of GST, wild-type GST-SHP-1, GST-SHP-1 lacking both SH2 domains (ΔN+CSH2), and GST-SHP-1 lacking only the N-terminal SH2 domain (ΔNSH2). Bound His-GIV-CT was analyzed by immunoblotting with GIV-CT pAb. His-GIV-CT specifically bound wild-type GST-SHP-1 but not GST alone (lanes 3 and 4); binding was reduced in the absence of both SH2 domains (lane 5) but restored when the C-terminal SH2 domain was present (lane 6).

FIGURE 6.

WT, but not the catalytically inactive CS mutant of SHP-1, inhibits the formation of GIV-p85α(PI3K) complexes after EGF stimulation. COS7 cells transfected with vector alone (lanes 1–3), HA-SHP-1 WT (lane 4), or HA-SHP-1 CS mutant (lane 5) were serum-starved (−) and subsequently treated with 50 nm EGF before lysis. Equal aliquots of lysates (bottom) were incubated with preimmune (lane 1) or anti-GIV-CT (lanes 2–5) IgGs and protein A-agarose beads. Immune complexes (top) were analyzed for endogenous GIV and p85α by immunoblotting (IB). p85α was detectable in GIV-bound complexes after EGF stimulation but not in starved cells (compare lanes 2 and 3), undetectable in cells expressing SHP-1 WT but restored in cells expressing SHP-1 C453S. Ab, antibody.

FIGURE 7.

SHP-1 inhibits the GIV ability to enhance Akt phosphorylation. A and B, WT, but not the catalytically inactive CS mutant of SHP-1 inhibits GIV-dependent Akt phosphorylation. A, COS7 cells were transfected with empty vector (lane 1), GIV-FLAG alone (lane 2), GIV-FLAG and HA-SHP-1 WT (lane 3), or GIV-FLAG and HA-SHP-1 CS (lane 4). Cells were maintained in the presence of 2% FBS for 20 h before lysis. Equal aliquots of whole cell lysates were analyzed for FLAG, HA (HA-SHP-1), phospho-Akt (pAkt), actin, and tubulin by immunoblotting. Akt phosphorylation at Ser-473 (pAkt) was increased in cells expressing GIV-FLAG (compare lanes 1 and 2), decreased in cells co-expressing GIV-FLAG and SHP-1 WT (lane 3), and increased in cells co-expressing GIV-FLAG and SHP-1 CS (lane 4). B, bar graphs showing quantification of pAkt:actin ratios in A expressed as -fold increase compared with vector control. Quantifications were performed by band densitometry using LiCOR Odyssey Infrared Imager. Results are shown as the mean ± S.E. (n = 5). C, depletion of SHP-1 enhances Akt phosphorylation at 5 min after EGF stimulation. HeLa cells treated with scrambled (lanes 1–3) or SHP-1 (lanes 4 and 5) siRNA were serum-starved and subsequently stimulated with EGF for 5 and 15 min as in Fig. 2C. Equal aliquots of whole cell lysates were analyzed for phosphorylated Akt, phosphorylated ERK (pERK1/2), SHP-1, and tubulin by immunoblotting (IB). pAkt, but not pERK1/2, was specifically and significantly enhanced at both 5 and 15 min after EGF stimulation in SHP-1-depleted cells as compared with control cells (compare lanes 4 and 5 with 2 and 3), temporally coinciding with the enhancement of tyrosine phosphorylation of GIV observed in Fig. 2C.

FIGURE 8.

Summary and working model. Tyrosine phosphorylation of GIV is triggered by ligands for either RTKs (i.e. EGFR) or GPCRs (i.e. LPA receptor). Activation of non-RTKs (i.e. Src) downstream of both classes of receptors can also phosphorylate GIV on identical tyrosines (14). Tyrosine-phosphorylated GIV directly binds p85α and activates Class 1 PI3Ks, which in turn activate Akt. SHP-1 protein-tyrosine phosphatase is activated downstream of both RTKs and GPCRs. Activated SHP-1 is known to bind and dephosphorylate the autophosphorylation site(s) on the cytoplasmic tail of EGFR (19, 20) and is known to effectively dephosphorylate substrates of Src kinase (56). Here we demonstrate that SHP-1 binds and dephosphorylates GIV, prevents the formation of GIV-p85α(PI3K) complexes, and thereby inhibits activation of Akt via the GIV-PI3K axis.