Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase.
The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) 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, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that 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 with PTX. These findings show that Gi 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 Gi enables 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 intermediates in the p42/p44 MAPK cascade.
Conway
A M
AM
Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, Scotland, U.K.
Rakhit
S
S
Pyne
S
S
Pyne
N J
NJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006168:Guinea Pigs; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008954:Models, Biological; D009130:Muscle, Smooth / Q000378:metabolism*; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D012137:Respiratory System / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism
D010982:Platelet-Derived Growth Factor; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go