Sphingosylphosphorylcholine rapidly induces tyrosine phosphorylation of p125FAK and paxillin, rearrangement of the actin cytoskeleton and focal contact assembly. Requirement of p21rho in the signaling pathway.
Sphingosylphosphorylcholine (SPC), a potent mitogen for Swiss 3T3 cells, rapidly induced tyrosine phosphorylation of multiple substrates including bands of M(r) 110,000-130,000 and M(r) 70,000-80,000 in Swiss 3T3 cells. Focal adhesion kinase (p125FAK) and paxillin were identified as prominent substrates for SPC-stimulated tyrosine phosphorylation. An increase in tyrosine phosphorylation of p125FAK was detected as soon as 30 s after SPC stimulation, reaching a maximum after 2.5 min. SPC induced tyrosine phosphorylation of p125FAK in a concentration-dependent fashion; a half-maximum effect occurred at 250 nM. Tyrosine phosphorylation of p125FAK induced by SPC could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. SPC induced a unique pattern of reorganization of the actin cytoskeleton with a rapid appearance of actin microspikes at the plasma membrane that was followed by the formation of actin stress fibers. This pattern of cytoskeletal changes was clearly distinguishable from that induced by bombesin and 1-oleoyl-lysophosphatidic acid. Formation of microspikes and actin stress fibers were accompanied by striking assembly of focal adhesion plaques. Cytochalasin D, which disrupts the network of actin microfilaments, completely prevented SPC-induced tyrosine phosphorylation of p125FAK. In addition, tyrosine phosphorylation of p125FAK was markedly inhibited in the presence of platelet-derived growth factor at a concentration (30 ng/ml) that disrupts actin stress fibers. Finally, microinjection of Clostridium botulinum C3 exoenzyme, which inactivates p21rho, prevented SPC-induced formation of actin stress fibers, focal adhesion assembly, and tyrosine phosphorylation. Thus, p21rho is upstream of both cytoskeletal reorganization and tyrosine phosphorylation in SPC-treated cells.
Seufferlein
T
T
Imperial Cancer Research Fund, London, United Kingdom.
Rozengurt
E
E
D016475:3T3 Cells; D036002:ADP Ribose Transferases / Q000008:administration & dosage / Q000494:pharmacology; D000199:Actins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001839:Bombesin / Q000494:pharmacology; D001905:Botulinum Toxins; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008845:Microinjections; D051419:Paxillin; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020741:rho GTP-Binding Proteins
D000199:Actins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D019000:Phosphotyrosine; D015240:Phorbol 12,13-Dibutyrate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C070515:bisindolylmaleimide I; D013110:Sphingosine; D001839:Bombesin; D002118:Calcium