Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxinsensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase

The mechanism used by the platelet-derived growth factor receptor (PDGFR) t o activate the mitogen-activated- protein kinase (p(42)/p(44) MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have fou nd that pertussis toxin (PTX, which was used to inactivate the heterotrimer ic G-protein G(i)) induced an approx. 40-50 % decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PPI, which abolished p42/p44 MAPK activation (PP I and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermo re, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeare d correlated. These findings suggest GFR can utilize the PTX-sensitive G-pr otein, G(1), to regulate c-Src and subsequent p42/p44 MAPK( activation. Pho sphoinositide 3-kinase (PI3K) has been shown by others to be involved in p4 2/p44 MAPK activation. This is confirmed here by experiments which showed t hat PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42 /p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells wit h PTX. These findings show that G(i) might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that G(i) ena bles the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are i ntermediates in the p42/p44 MAPK cascade.