Dual regulation of sphingosine 1-phosphate-induced phospholipase D activity through RhoA and protein kinase C-alpha in C2C12 myoblasts

Previous studies showed that in C2C12 cells, phospholipase D (PLD) and its known regulators, RhoA and protein kinase C alpha (PKC alpha), were downstr eam effectors in sphingosine I-phosphate (SPP) signalling. Moreover, the ro le of PKC for SPP-mediated PLD activation and the requirement of PKC alpha for RhoA translocation were reported. The present results demonstrated that inactivation of RhoA, by overexpression of RhoGDP dissociation inhibitor ( RhoGDI) as well as treatment with C3 exotoxin, attenuated SPP-stimulated PL D activity, supporting the involvement of RhoA in the stimulation of PLD ac tivity by the bioactive lipid in C2C12 myoblasts. In addition, the effect o f PKC alpha inhibitor Go6976 on the SPP-induced PLD activation in myoblasts , where RhoA function was inactivated, was consistent with a dual regulatio n of the enzyme through RhoA and PKCa. Interestingly, the subcellular distr ibution of PLD isoforms, RhoA and PKC alpha, in SPP-stimulated cells suppor ted the view that the functional relationship between the two PLD regulator s, demonstrated to occur in SPP signalling, represents a novel mechanism of regulation of specifically localized PLD. (C) 2001 Elsevier Science Inc. A ll rights reserved.