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 [3H]phosphatidylbutanol ([3H]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 Ro-31-8220 completely abolished the generation of [3H]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 [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM 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.
Briscoe
C P
CP
University of Birmingham Institute for Cancer Studies, Queen Elizabeth Hospital, Birmingham, U.K.
Martin
A
A
Cross
M
M
Wakelam
M J
MJ
D016475:3T3 Cells; D000818:Animals; D001839:Bombesin / Q000494:pharmacology*; D019833:Genistein; D020404:Glycerophospholipids; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D020404:Glycerophospholipids; D007211:Indoles; D007529:Isoflavones; D010712:Phosphatidic Acids; C076715:pervanadate; C068564:phosphatidylbutanol; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014638:Vanadates; D006160:Guanosine Triphosphate; D019833:Genistein; D011493:Protein Kinase C; D017027:Protein Tyrosine Phosphatases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D001839:Bombesin; C064758:Ro 31-8220