THE ROLES OF MULTIPLE PATHWAYS IN REGULATING BOMBESIN-STIMULATED PHOSPHOLIPASE-D ACTIVITY IN SWISS 3T3 FIBROBLASTS

The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [H-3]phosphatidylbutanol ([H-3]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx, 50 % by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Re-31-8220 completely abolished the generation of[H-3]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [H-3]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 mu M GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.