Protein kinase C-theta (PKCθ) phosphorylates and inhibits the guanine exchange factor, GIV/Girdin

Abstract

Gα-interacting, vesicle-associated protein (GIV/Girdin) is a multidomain signal transducer that enhances PI3K-Akt signals downstream of both G-protein-coupled receptors and growth factor receptor tyrosine kinases during diverse biological processes and cancer metastasis. Mechanistically, GIV serves as a non-receptor guanine nucleotide exchange factor (GEF) that enhances PI3K signals by activating trimeric G proteins, Gαi1/2/3. Site-directed mutations in GIV's GEF motif disrupt its ability to bind or activate Gi and abrogate PI3K-Akt signals; however, nothing is known about how GIV's GEF function is regulated. Here we report that PKCθ, a novel protein kinase C, down-regulates GIV's GEF function by phosphorylating Ser(S)1689 located within GIV's GEF motif. We demonstrate that PKCθ specifically binds and phosphorylates GIV at S1689, and this phosphoevent abolishes GIV's ability to bind and activate Gαi. HeLa cells stably expressing the phosphomimetic mutant of GIV, GIV-S1689→D, are phenotypically identical to those expressing the GEF-deficient F1685A mutant: Actin stress fibers are decreased and cell migration is inhibited whereas cell proliferation is triggered, and Akt (a.k.a. protein kinase B, PKB) activation is impaired downstream of both the lysophosphatidic acid receptor, a G-protein-coupled receptor, and the insulin receptor, a receptor tyrosine kinase. These findings indicate that phosphorylation of GIV by PKCθ inhibits GIV's GEF function and generates a unique negative feedback loop for downregulating the GIV-Gi axis of prometastatic signaling downstream of multiple ligand-activated receptors. This phosphoevent constitutes the only regulatory pathway described for terminating signaling by any of the growing family of nonreceptor GEFs that modulate G-protein activity.

Fig. 1.

GIV cannot bind or activate Gαi3 when Ser¹⁶⁸⁹ in its GEF motif is mutated to Asp. (A) Diagram showing the phylogenetically conserved sequence of the GEF motif of GIV. Sequences obtained from the accession numbers (in brackets) were aligned using Clustal W. The conserved Phe¹⁶⁸⁵ (F1685, green), previously identified as the key residue that establishes a hydrophobic interaction with Trp²¹¹ and Phe²¹⁵ of Gαi3 (6), and the predicted phosphoregulated Ser¹⁶⁸⁹ (yellow) are shown. Conserved residues are shaded in black and similar residues in gray. (B) Homology model of Gαi3 in complex with GIV 1678–1689 generated (6) using the structure of the synthetic peptide KB-752 bound to Gαi1 [Protein Data Bank ID 1Y3A] as a template (32). Yellow, Gαi3 subunit; red, GIV’s GEF motif; white spheres, hydrogen; red spheres, oxygen; blue sphere, nitrogen. Ser¹⁶⁸⁹ lies in close proximity to the Gαi-GIV interface. (C) GIV fails to coimmunoprecipitate with Gαi3 when Ser¹⁶⁸⁹ in GIV is mutated to Asp. Immunoprecipitation was carried out with anti-FLAG mAb on equal aliquots of Cos7 lysates coexpressing FLAG-Gαi3 and GIV-WT, SD, or FA followed by incubation with protein-G beads. Lysates (Lower) and bound immune complexes (Upper) were analyzed for FLAG (Gαi3-FLAG), GIV, and Gβ by immunoblotting (IB). Binding of GIV-SD (lane 3) or FA (lane 4) mutants to Gαi3-FLAG is dramatically reduced compared with that of GIV-WT (lane 2), whereas Gβ binds equally under all conditions. (D) Mutation of Ser¹⁶⁸⁹ in GIV to Asp [S1689→D (SD)] virtually abolishes binding of His-GIV-CT to GST-Gαi3. Equal aliquots (1.2 μg) of WT (lanes 1 and 2), SD (lane 3), or SA (lane 4) His-GIV-CT proteins were incubated with 5 μg GST or GST-Gαi3 preloaded with GDP and immobilized on glutathione beads. Bound proteins (Top) were analyzed by IB for His (GIV-CT). Equal loading of GST and His-GIV-CT proteins was confirmed by Ponceau S staining (Middle) and by immunoblotting for His (inputs, Lower). (E) Activation of His-Gαi3 by His-GIV-CT is virtually abolished in the presence of the GIV-SD (open circles) mutant compared with GIV-WT (solid circles). The steady-state GTPase activity of His-Gαi3 (50 nM) was determined in the presence of purified His-GIV-CT-WT and SD. Gαi3 activation is expressed as percent of the steady-state GTPase activity of Gαi3 alone. Results are shown as mean ± SEM of three experiments.

Fig. 2.

GIV is phosphorylated on Ser¹⁶⁸⁹ by PKCθ. (A) In vitro kinase assays were carried out with recombinant PKA, PKB, and rat brain PKC and equal aliquots (2 μg) of either WT (lane 1) or a nonphosphorylatable SA His-GIV-CT mutant (lane 2). Phosphorylated proteins were detected by autoradiography (Upper three panels), and equal loading of His-GIV-CT substrates was confirmed by Coomassie Blue staining (Lower panel). Although PKA, PKB, and PKC phosphorylate GIV-CT in vitro, Ser¹⁶⁸⁹ is phosphorylated exclusively by PKC. (B) In vitro kinase assays were carried out with 100 ng of novel PKC isoforms and analyzed by autoradiography. The theta (θ) isoform of PKC specifically phosphorylates GIV at Ser¹⁶⁸⁹. (C) PKCθ-mediated phosphorylation of GIV at Ser¹⁶⁸⁹ abolishes the ability of His-GIV-CT to bind GST-Gαi3. Equal aliquots (2 μg) of WT or SA His-GIV-CT were either mock treated (lanes 1, 2, and 4) or phosphorylated with 100 ng PKCθ (lanes 3 and 5) before their use in GST pulldown assays with 10 µg control GST (lane 1) or 5 µg GST-Gαi3 (lanes 2–5) preloaded with GDP and immobilized on glutathione beads. Bound proteins (Upper) and inputs (Lower) were analyzed by immunoblotting (IB) for His (His-GIV-CT).

Fig. 3.

PKCθ is required for phosphorylation of GIV at Ser¹⁶⁸⁹ after ligand stimulation. HeLa cells treated with scrambled (Scr) or PKCθ siRNA were serum starved (lanes 1 and 2) and stimulated with serum (lanes 3 and 4), PMA (10 min; lanes 5 and 6), insulin (5 min; lanes 7 and 8), EGF (5 min; lanes 9 and 10), or LPA (20 min; lanes 11 and 12) before lysis. (Upper) Equal aliquots of lysates were analyzed for phospho-Ser1689 GIV (pS1689 GIV), total GIV (GIV-CT Ab), PKCθ, and tubulin by quantitative immunoblotting (IB). (Lower) Bar graphs show the ratio of pS1689-GIV to total GIV, normalized to control siRNA-treated HeLa cells within each condition. Results shown are representative of five independent experiments. The concentration and duration of stimulation in each case are based on the peak activation of Akt via GIV’s GEF motif observed previously (2, 5, 6, 8, 12).

Fig. 4.

PKCθ binds and colocalizes with GIV at the cell periphery after ligand stimulation. (A) GIV coimmunoprecipitates with YFP-PKCθ from lysates of cells stimulated with PMA (lane 3) or insulin (lane 4), but not from serum-starved cells (lane 2). Cos7 cells expressing YFP-PKCθ were serum starved (0% FBS, 16 h) and then stimulated with either PMA (200 nM, 10 min; lane 3) or insulin (100 nM, 5 min; lane 4) before lysis. Immunoprecipitation was carried out with anti-GFP mAb (lanes 2–4) or control IgG (lane 1) from equal aliquots of lysates (Lower), followed by incubation with protein-G agarose beads. Bound immune complexes (Upper) were analyzed for GIV (CT Ab) and GFP (YFP-PKCθ) by immunoblotting (IB). (B) Endogenous PKCθ and GIV partially colocalize at the cell periphery after ligand stimulation. In starved HeLa cells endogenous PKCθ is predominantly cytosolic, whereas GIV is predominantly located at the perinuclear Golgi region as demonstrated previously (7, 33). After stimulation with EGF (50 nM) or insulin (100 nM), small but significant pools of GIV and PKCθ were seen in patches at the PM (arrowheads, black and white single panels) by confocal microscopy. Yellow pixels in the merged images at the PM denote patchy colocalization (arrowheads) of GIV (red) and PKCθ (green) in stimulated cells, but not in starved cells. (Scale bar, 10 μm.)

Fig. 5.

Phosphorylation of GIV at Ser¹⁶⁸⁹ inhibits Akt activation, actin stress fiber formation, and cell migration and enhances cell proliferation. (A and B) HeLa cell lines stably expressing siRNA-resistant GIV-WT-FLAG (HeLa GIV-WT), GIV-FA-FLAG (HeLa GIV-FA), or GIV-SD-FLAG (HeLa GIV-SD) were treated with control (Scr) or GIV siRNA for 36 h, serum starved [0.2% (vol/vol) FBS, 16 h], and then stimulated with insulin for 5 min (A) or LPA for 20 min (B) before lysis. Whole-cell lysates were analyzed for phospho Akt (pAkt) (S473), total Akt (tAkt), and tubulin by quantitative immunoblotting (immunoblots shown in Fig. S5 B and C). Bar graphs display the ratio of pAkt/tAkt (y axis), normalized to starved control HeLa cells. Results are shown as mean ± SD of three independent experiments. (C) All three HeLa-GIV stable cell lines were depleted of endogenous GIV, fixed, and costained with phalloidin-Texas Red (F-actin, red) and DAPI/DNA (blue) and visualized by fluorescence. (Scale bar, 10 μm.) (D) HeLa cell lines were treated with scrambled (Scr) or GIV siRNA as indicated, grown to 100% confluency as monolayers, scratch wounded, and assessed for wound closure by serial imaging of the wound for 12 h (see SI Experimental Procedures). Bar graphs show quantification of the percentage of wound area closed by 12 h, which is expressed as migration index (y axis). (E and F) HeLa cells grown on coverslips were fixed and costained with DAPI/DNA (blue) and either phospho-Histone H3 (E) or anti-BrdU mAb (F). Bar graphs display the proliferation rate, i.e., expressed as mitotic index (y axis) derived by quantifying percentage of phospho-Histone- or BrdU-positive cells (see SI Experimental Procedures). Images of representative fields are shown in Fig. S5 D and E. Results are shown as mean ± SD of 8–12 fields. P values: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Proposed model for how GIV’s GEF function is regulated by PKCθ. Upon ligand stimulation of RTKs and GPCRs GIV is recruited to the PM (2, 7), where GIV’s GEF motif activates Gi and releases free Gβγ subunits, which in turn activate Akt via class 1 PI3Ks (6). Gβγ is also known to activate PLC (18), as can multiple other pathways downstream of RTKs and GPCRs (dashed arrows). Once activated, PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG) (17). The latter is a potent stimulus for recruitment and activation of PKCθ. Activated PKCθ binds and phosphorylates GIV at Ser¹⁶⁸⁹. This phosphoevent inhibits GIV’s ability to bind and activate Gi, thereby down-regulating GIV-Gi-dependent cellular processes. Thus, PKCθ-dependent phosphorylation of GIV serves as a negative feedback loop (dashed line) that terminates the GIV-Gi pathway of signaling.