Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLC gamma

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mito genic response of platelet-derived growth factor (PDGF) and is formed by ac tivation of sphingosine kinase. We examined the effect of PDGF on sphingosi ne kinase activation in TRMP cells expressing wild-type or various mutant b eta PDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expr essing wild-type receptors but not in cells expressing kinase-inactive rece ptors (R634). Cells expressing mutated PDGF receptors with phenylalanine su bstitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1 021 (F5 mutants), which are unable to associate with PLC gamma, phosphatidy linositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phos phatase SHP-2, not only failed to increase DNA synthesis in response to PDG F but also did not activate sphingosine kinase. Moreover, mutation of tyros ine-1021 of the PDGF receptor to phenylalanine, which impairs its associati on with PLC gamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, respons ible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 rece ptor 'add-back' mutants in which association with the Pas GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored . However, a mutant PDGF receptor that was able to bind PLC gamma (tyrosine -1021), but not other signaling proteins, restored sphingosine kinase sensi tivity to PDGF. These data indicate that the tyrosine residue responsible f or binding of PLC gamma is required for PDGF-induced activation of sphingos ine kinase. Moreover, calcium mobilization downstream of PLC gamma, but not protein kinase C activation, appears to be required for stimulation of sph ingosine kinase by PDGF.