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

Abstract

Sphingosylphosphorylcholine (SPC), a potent mitogen for Swiss 3T3 cells, rapidly induced tyrosine phosphorylation of multiple substrates including bands of Mr 110,000-130,000 and Mr 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.