Brian E. Hawes

Role of c-Src Tyrosine Kinase in G Protein-coupled Receptorand Gβγ Subunit-mediated Activation of Mitogen-activated Protein Kinases

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gβγ subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc·Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gβγ subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gβγ subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gβγ subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc·Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gβγ subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc·Grb2 complex formation, and MAP kinase activation. These data suggest that Gβγ subunit-mediated formation of Shc·c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.

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

The βγ-subunit of Gi 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 Gβγ mediates tyrosine phosphorylation of Shc, however, is unclear. This study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in Gβγ-mediated MAP kinase activation. We show that Gi-coupled receptor- and Gβγ-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 Gi-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 Gβγ-mediated MAP kinase signaling pathway at a point upstream of Sos and Ras activation.

Regulation of Mitogen-Activated Protein Kinase Pathways by Catecholamine Receptors

Publisher Summary Until recently little has been known about the mechanisms of mitogenic signal transduction employed by receptors that couple to heterotrimeric G-proteins (GRs). These receptors participate in the regulation of cell proliferation in both physiological and pathophysiological states, and in cellular transformation in some, mostly neuroendocrine, human tumors. GRs that mediate cellular responses to a variety of humoral, endothelium-, or platelet-derived substances have been found to rapidly stimulate the mitogen-activated protein kinase (ERK1/2) pathway, a major point of convergence for signals regulating cell growth and differentiation. The best understood pathway of ERK1/2 activation is that mediated by growth factor receptors that possess intrinsic ligand-stimulated tyrosine kinase activity (RTK), such as the receptor for epidermal growth factor (EGF). To understand the mechanisms whereby GRs mediate growth regulatory signals, the mechanisms whereby several GRs, including the α 1 B AR and α 2 A AR, stimulate ERK1/2 activity in transiently transfected COS-7 and Chinese hamster ovary (CHO) cell model systems has been studied. In these cells, clear heterogeneity exists between the mechanisms of ERK1/2 activation employed by receptors that signal via pertussis toxin-sensitive G i family proteins, such as α 2 A AR, and receptors that signal via pertussis toxininsensitive G q/11 family proteins, such as α 1 B AR.