A function for phosphoinositide 3-kinase β lipid products in coupling βγ to Ras activation in response to lysophosphatidic acid

Abstract

Although Gβγ is thought to mediate mitogen-activated protein kinase (MAPK) activation in response to G protein-coupled receptor stimulation, the mechanisms involved in this pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K) has been proposed as an early intermediate in this process, but its role has remained elusive. We have observed that dominant negative mutants of p110β, but not of p110γ, inhibited MAPK stimulation in response to lysophosphatidic acid (LPA). The role of p110β was located upstream from Ras. To determine which of the lipid or protein kinase activities of p110β were important for Ras activation, we produced a mutant p110β lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110β lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to specifically inhibit Ras stimulation induced by LPA. In addition, we have observed that the PH domain-containing adapter protein Gab1, which is involved in p110β activation during LPA stimulation, is also implicated in this pathway downstream of p110β. Indeed, both membrane redistribution and phosphorylation of Gab1 were reduced in the presence of PI3K inhibitors or dominant negative p110β. Downstream of Gab1, the tyrosine phosphatase SHP2 was found to mediate Ras activation in response to LPA and to be recruited through PI3K and Gab1, because transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras activation without interfering with PI3K activation. We conclude that LPA-induced Ras activation is mediated by a p110β/Gab1/SHP2 pathway. Moreover, we present data indicating that p110β is effectively the target of βγ in this pathway, suggesting that the p110β/Gab1/SHP2 pathway provides a novel link between βγ and Ras by integrating two early events of LPA signaling, i.e. Gβγ release and tyrosine kinase receptor transactivation.