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.
Highlights
Recent work has provided mechanistic insights into these biological functions of GIV
Src homology 2-containing phosphatase-1 (SHP-1) Dephosphorylates GIV/Girdin 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 receptor-tyrosine kinases (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 plasma membrane (PM), which in turn triggers recruitment and activation of Akt
We previously demonstrated [14] that ligand stimulation of either RTKs or G-protein coupled receptors (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
Summary
Recent work has provided mechanistic insights into these biological functions of GIV. 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).
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