SPHINGOSYLPHOSPHORYLCHOLINE ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE IN SWISS 3T3 CELLS REQUIRES PROTEIN-KINASE-C AND A PERTUSSIS-TOXIN-SENSITIVE G-PROTEIN

Sphingosylphosphorylcholine (SPC) is a potent mitogen for Swiss 3T3 cells, but the signaling mechanisms involved are poorly characterized, Here, we report that addition of SPC induces a rapid and transient activation of p42 mitogen-activated protein kinase (p42(MAPK)) in these cells, SPC induced p42(MAPK) activation peaked at 5 min and was undetectable after 30 min of incubation with SPC. The effect of SPC on p42(MAPK) activation was comparable to that induced by bombesin and platelet-derived growth factor. As SPC strongly induced phosphorylation of the major protein kinase C (PKC) substrate 80W/MARCKS in either intact or permeabilized cells, we examined whether PKC could be involved in SPC-induced p42(MAPK) activation. Here, we demonstrate that p42(MAPK) activation by SPC was dependent on PKC activity as shown by inhibition of PKC with the bisindolylmaleimide GF 109203X or down-regulation of PKC by prolonged treatment of Swiss 3T3 cells with phorbol esters, Activation of both PKC and p42(MAPK) by SPC was markedly inhibited by treatment with pertussis toxin, implicating a G protein(s) of the G(i)/G(o) subfamily in the action of SPC. SPC-induced rapid activation of a downstream target of p42(MAPK), p90 ribosomal S6 kinase (p90(rsh)), also required PKC and a pertussis toxin-sensitive G protein, In addition, SPC-induced mitogenesis was dependent on a G(i) protein in Swiss 3T3 cells, SPC also induced p42(MAPK) activation and DNA synthesis in secondary cultures of mouse embryo fibroblasts through a pertussis toxin-sensitive pathway, As G proteins link many cell surface receptors to effector proteins, we hypothesize, therefore, that SPC could bind to a receptor that mediates at least some of its biological effects in Swiss 3T3 cells and mouse embryo fibroblasts.