GIV is a nonreceptor GEF for G alpha i with a unique motif that regulates Akt signaling

Abstract

Heterotrimeric G proteins are molecular switches that control signal transduction. Ligand-occupied, G protein-coupled receptors serve as the canonical guanine nucleotide exchange factors (GEFs) that activate heterotrimeric G proteins. A few unrelated nonreceptor GEFs have also been described, but little or nothing is known about their structure, mechanism of action, or cellular functions in mammals. We have discovered that GIV/Girdin serves as a nonreceptor GEF for G alpha i through an evolutionarily conserved motif that shares sequence homology with the synthetic GEF peptide KB-752. Using the available structure of the KB-752 x G alpha i1 complex as a template, we modeled the G alpha i-GIV interface and identified the key residues that are required to form it. Mutation of these key residues disrupts the interaction and impairs Akt enhancement, actin remodeling, and cell migration in cancer cells. Mechanistically, we demonstrate that the GEF motif is capable of activating as well as sequestering the G alpha-subunit, thereby enhancing Akt signaling via the G betagamma-PI3K pathway. Recently, GIV has been implicated in cancer metastasis by virtue of its ability to enhance Akt activity and remodel the actin cytoskeleton during cancer invasion. Thus, the novel regulatory motif described here provides the structural and biochemical basis for the prometastatic features of GIV, making the functional disruption of this unique G alpha i-GIV interface a promising target for therapy against cancer metastasis.

Fig. 1.

Identification of a putative novel motif in GIV (amino acid 1623–1870) responsible for the preferential binding to GDP·Gαi3. (A) The indicated in vitro translated ³⁵S-Met labeled GIV constructs were incubated with approximately 15 μg GST–Gαi3 or GST preloaded with GDP or GDP·AlF₄⁻ immobilized on glutathione beads. Bound proteins were analyzed by autoradiography, and equal loading of GST proteins was confirmed by protein staining (data not shown). (B) A phylogenetically conserved sequence in the C terminus of GIV (amino acid 1678–1694, “GEF motif”) shows similarity to the synthetic peptide KB-752. Sequences obtained from the accession numbers (Fig. S1) were aligned using Clustal W. Conserved residues are shaded in black, similar residues in gray.

Fig. 2.

Identification of the critical residues implicated in forming the interface between Gαi3 and the novel interacting motif in GIV. (A) Homology model showing the critical residues forming the predicted interface between Gαi3 and the conserved motif found in GIV. Model was generated by the Swiss-Model server using the KB-752·Gαi1 structure [PDB: 1Y3A] as template. (B) F1685A and E1688L mutations in GIV abolish its binding to Gαi3. In vitro translated ³⁵S-Met labeled GIV or the indicated GIV mutants were incubated with approximately 15 μg GST-Gαi3 or GST preloaded with GDP or GDP·AlF₄⁻ immobilized on glutathione beads. Bound proteins were analyzed by autoradiography. (C) R208L, W211A, and F215A mutations in Gαi3's switch II region impair binding to GIV. COS-7 cell lysates were incubated with GST–Gαi3 or the indicated mutants as in (B). Bound proteins were analyzed by immunoblotting for GIV. Equal loading of GST proteins in (B) and (C) was confirmed by protein staining (data not shown).

Fig. 3.

GIV is a guanine nucleotide exchange factor for Gαi subunits; (A) His–GIV–CT (1 μM, open circles) but not His–GIV–CT F1685A (1 μM, open triangles) increases the steady-state GTPase activity of His–Gαi3 over His–Gαi3 alone (50 nM, closed circles). Parallel experiments were run in the absence of His–Gαi3 (dotted lines). (B) His–GIV–CT and the KB-752 peptide increase the steady-state GTPase activity of His–Gαi3 in a dose-dependent manner. The amount of GTP hydrolyzed in 10 min by His–Gαi3 was determined in the presence of the indicated amounts of His–GIV–CT or KB-752 peptide and the results were fitted to a sigmoidal curve.

Fig. 4.

GIV enhances Akt signaling via Gβγ-dependent activation of PI3K. (A and B) Inhibition of PI3K and Gβγ signaling blocks GIV-induced Akt phosphorylation. COS-7 cells were transfected with the indicated plasmid constructs and 48 h later incubated with DMSO or 10 μM LY294002 (PI3K inhibitor) for 2 h (A), 10 μM gallein (Gβγ “hotspot” inhibitor), or 10 μM fluorescein (negative control for gallein) for 30 min (B). Cells were lysed and analyzed by immunoblotting (IB). These experiments were performed in the presence of 10% FBS. (C) Structural model of (Left) the GEF motif of GIV bound to Gαi3 described in Figs. 2A and S2 compared to (Right) Gβ-subunit bound to Gαi1 (24). Blue: Gα subunit, Green: Switch II region, Red: “GEF motif” of GIV or Gβ. His–GIV–CT (D) but not His–GIV–CT F1685A (E) displaces Gβγ from GST–Gαi3 (approximately 7 μg). Equal amounts of preformed GST–Gαi3·Gβγ complexes immobilized on glutathione beads were incubated with increasing amounts of His–GIV–CT, and bound proteins were analyzed by immunoblotting (IB) with the indicated antibodies. Equal loading of GST proteins was confirmed by protein staining (data not shown).

Fig. 5.

Role of GIV's GEF motif on Akt activation and tumor cell migration. Depletion of endogenous GIV by siRNA impairs Akt activation (pAkt) upon LPA (A) or insulin (B) stimulation in HeLa cells stably expressing siRNA-resistant GIV F1685A (HeLa–GIV FA) but not in HeLa cells expressing siRNA resistant wt GIV (HeLa–GIV WT). The different HeLa cell lines were treated with GIV siRNA or control (Scr) siRNA oligos, serum starved for 6 h, and then stimulated with 10 μM LPA for 20 min (A) or 100 nM insulin for 5 min (B). Cell lysates were analyzed by immunoblotting (IB). (C) Only HeLa–GIV WT cells migrated efficiently in scratch-wound assays upon depletion of endogenous GIV. Cell migration was determined as described in Materials and Methods after treatment with GIV or control (Scr) siRNA oligos. Endogenous GIV expression was reduced approximately 75% upon siRNA treatment. (D) HeLa–GIV FA cells show absence of actin stress fibers compared to HeLa–GIV WT cells. HeLa–GIV WT and HeLa–GIV FA cells were co-stained with phalliodin-Texas red (F-actin, red) and DAPI (DNA, blue) and visualized by fluorescence. (E and F) MCF7 cells stably expressing GIV (MCF7-GIV WT) but not those expressing F1685A (MCF7-GIV FA) showed enhanced migration in scratch-wound assays (E) and enhanced Akt phosphorylation (F) compared to MCF7 cells expressing the vector control (MCF7-V). *** = P < 0.001 compared to vector control; ### = P < 0.001 compared to cells transfected with GIV WT.