Focal adhesion kinase (p125FAK) and paxillin are substrates for sphingomyelinase-induced tyrosine phosphorylation in Swiss 3T3 fibroblasts.
We examined the effect of sphingomyelinase on tyrosine phosphorylation of intracellular proteins in mouse Swiss 3T3 fibroblasts. Incubation of the cells with bacterial sphingomyelinase resulted in the elevation of tyrosine phosphorylation of multiple cellular proteins of 190, 130, 120, 97 and 70 kDa within minutes. The 120 and 70 kDa tyrosine-phosphorylated peptides were identified as p125 focal adhesion kinase (p125FAK) and paxillin respectively by the use of specific antibodies against the proteins. Tyrosine kinase activity associated with anti-p125FAK immunoprecipitate was stimulated by incubation of cells with sphingomyelinase. Cytochalasin D, which selectively disrupts the network of actin filaments, inhibited sphingomyelinase-induced tyrosine phosphorylation of p125FAK and elevation of tyrosine kinase activity in the anti-p125FAK immunoprecipitates. Sphingomyelinase-induced phosphorylation of p125FAK was not inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. This was in sharp contrast with a wortmannin-sensitive phosphorylation of p125FAK observed in platelet-derived growth factor (PGDF)-stimulated cells. Thus hydrolysis of sphingomyelin is considered to regulate the tyrosine kinase cascade including p125FAK and paxillin by a mechanism distinct from PDGF.
Sasaki
T
T
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hazeki
K
K
Hazeki
O
O
Ui
M
M
Katada
T
T
D016475:3T3 Cells; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051379:Mice; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D015815:Cell Adhesion Molecules; D002518:Ceramides; D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; C496062:Pxn protein, mouse; D015638:Cytochalasin D; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D013108:Sphingomyelin Phosphodiesterase; D000077191:Wortmannin