Bombesin, diacylglycerols, and phorbol esters rapidly stimulate the phosphorylation of an M(r) = 80,000 protein kinase C substrate in permeabilized 3T3 cells. Effect of guanine nucleotides

We have used digitonin permeabilization to study the mechanism of bombesin-induced activation of protein kinase C in Swiss 3T3 cells. Protein kinase C-mediated phosphorylations in permeabilized cells were identified using phorbol esters and diacylglycerols. Addition of phorbol 12,13-dibutyrate (PDBu) in the presence of [γ-³²P]ATP and digitonin caused a marked and rapid time- and dose-dependent increase in the phosphorylation of an M(r) 80,000 cellular protein (maximum stimulation = 12.6 ± 1.6-fold after 1 min, EC₅₀ = 27 nM). 1-oleoyl-2-acetylglycerol substituted for PDBu in stimulating the phosphorylation of M(r) 80,000 protein (EC₅₀ = 13 μM). Bombesin also caused a striking increase in the phosphorylation of M(r) 80,000 protein with a time course similar to that observed with PDBu. This phosphorylation was mimicked by mammalian bombesin-like peptides and blocked by the bombesin antagonists [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]substance P and [Leu¹³ψ(CH₂NH)Leu¹⁴]bombesin. Down-regulation of protein kinase C in intact cells by prolonged exposure to PDBu prevented M(r) 80,000 protein phosphorylation upon subsequent bombesin addition in digitonin-permeabilized cells.Comigration on one- and two-dimensional gel electrophoresis and phosphopeptide mapping confirmed that the M(r) 80,000 protein phosphorylated in permeabilized cells was indistinguishable from the M(r) 80,000 protein which is the major protein kinase C substrate in intact cells. The GDP analogue guanosine-5'-O-(2-thiodiphosphate) (GDPβS) caused a 70% inhibition of the bombesin-induced phosphorylation of M(r) 80,000 protein but had no effect on the phosphorylation induced by PDBu. Bombesin stimulated M(r) 80,000 protein phosphorylation in permeabilized cells in a dose-dependent manner (EC₅₀ = 4 nM), and GDPβS shifted the bombesin dose response curve to higher bombesin concentrations (EC₅₀ = 14 nM). These results demonstrate for the first time a growth factor receptor-mediated activation of protein kinase C in permeabilized cells and provide functional evidence for the involvement of a G protein in the transmembrane signaling pathway that mediates the stimulation of protein kinase C by bombesin in Swiss 3T3 cells.