Phosphatidylinositol 3-Kinase Is an Early Intermediate in the Gβγ-mediated Mitogen-activated Protein Kinase Signaling Pathway

Abstract

The ␤␥-subunit of G i mediates mitogen-activated protein (MAP) kinase activation through a signaling pathway involving Shc tyrosine phosphorylation, subsequent formation of a multiprotein complex including Shc, Grb2, and Sos, and sequential activation of Ras, Raf, and MEK. The mechanism by which G␤␥ mediates tyrosine phosphorylation of Shc, however, is unclear. This study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in G␤␥-mediated MAP kinase activation. We show that G i -coupled receptor-and G␤␥-stimulated MAP kinase activation is attenuated by the PI-3K inhibitors wortmannin and LY294002 or by overexpression of a dominant negative mutant of the p85 subunit of PI-3K. Wortmannin and LY294002 also inhibit G i -coupled receptor-stimulated Ras activation. The PI-3K inhibitors do not affect MAP kinase activation stimulated by overexpression of Sos, a constitutively active mutant of Ras, or a constitutively active mutant of MEK. These results demonstrate that PI-3K activity is required in the G␤␥-mediated MAP kinase signaling pathway at a point upstream of Sos and Ras activation.The cellular signaling pathways leading to receptor-tyrosine kinase- (RTK) 1 and G protein-coupled receptor-(GPCR) stimulated mitogen-activated protein (MAP) kinase activation have recently been the subject of intense investigation (1-6). The signaling pathway of RTK-mediated MAP kinase activation is the most clearly understood. Epidermal growth factor (EGF) stimulation, for example, produces activation and autophosphorylation of the EGF receptor leading to the formation of a multiprotein complex containing the phosphorylated receptor, the phosphoprotein Shc, the adaptor protein Grb2, and the Ras-GTP exchange factor Sos (7-9). Sos catalyzes exchange of GTP for GDP on the small guanine nucleotide-binding protein, Ras, thereby stimulating Ras activation (10). Ras-GTP activates a kinase cascade involving Raf, MEK, and MAP kinase (11-13). Activated MAP kinases phosphorylate and activate transcription factors involved in cell growth and proliferation (1). The signaling pathways utilized by G i -, G s -, G o -, and G qcoupled receptors to stimulate MAP kinase activation have also been assessed and compared (14 -18). In many cell types, G icoupled receptors mediate MAP kinase activation via the ␤␥-dependent activation of Ras (14 -16). Several of the intermediate steps in the G␤␥-stimulated MAP kinase pathway are identical with the RTK-stimulated signaling cascade including Shc phosphorylation, Shc/Grb2 association, and Sos activation (19). Inhibitors of Src family tyrosine kinase activity abrogate G i -coupled receptor-and G␤␥-mediated Shc phosphorylation and MAP kinase activation in COS-7 cells (3,17,20,21) suggesting that a Src family tyrosine kinase may be involved in the G␤␥-mediated MAP kinase activation pathway at a point upstream of Ras activation. The mechanism by which G␤␥ subunits mediate activation of a tyrosine kinase resulting in increased Shc phosphorylation, however, is unclear.Recent studies have suggested that phosph...

A specific function for phosphatidylinositol 3-kinase α (p85α-p110α) in cell survival and for phosphatidylinositol 3-kinase β (p85α-p110β) in de novo DNA synthesis of human colon carcinoma cells

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Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation

“…Incubation of cells with dBSA-LPA or FBS resulted in phosphorylation of endogenous Akt, and this activation was markedly inhibited by LY294002 or Wortmannin (Figure 4b). Because LPA can activate PI3K via the bg subunits of G i in COS cells 27 and Schwann cells, 28 we examined whether this pathway is activated by LPA treatment in HeLa cells. Phosphorylation of Akt by LPS was inhibited by pretreatment of HeLa cells with pertussis toxin (PTX) (Figure 4c).…”

Regulation of cell proliferation by G proteins

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