Inhibition of phosphoinositide hydrolysis and cell growth of Swiss 3T3 cells by myristoylated phospholipase C inhibitor peptides.

Abstract

It has been demonstrated that the phospholipase C-gamma (PLC-gamma) molecule contains within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of this region (PCI peptides) suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem. 267, 21884-21889]. In order to improve the permeability of the plasma membrane to PCI peptides, we synthesized myristoylated PCI peptides, myr-GLYRKAMRLRYPV [myr-PCI(Y)] and myr-GLFRKAMRLRFPV [myr-PCI(F)], which are identical except for the replacement of the two tyrosine residues in myr-PCI(Y) by phenylalanines in myr-PCI(F), and examined their inhibitory activity on PLC enzymes in vitro and in vivo. This fatty acid modification potentiated the inhibitory activity of the original PCI peptides and both myr-PCI(Y) and myr-PCI(F) suppressed the PIP2-hydrolyzing activity of purified PLC isoforms in vitro. The Ki values of myr-PCI(Y) and myr-PCI(F) for purified PLC-gamma1 were 3.5 and 55 microM, respectively. Myr-PCI(Y) at concentrations in the sub-micromolar range significantly suppressed IP3 formation induced by EGF, PDGF, bombesin, or serum in Swiss 3T3 cells. Furthermore, myr-PCI(Y) also strongly inhibited cell proliferation induced by these stimuli. The inhibitory effect on IP3 formation and proliferation of myr-PCI(F) was much less potent than that of myr-PCI(Y). These results suggest that myristoylated PCI peptides could be applied to living cells as specific inhibitors of PLC signaling pathways and that PLC pathways are at least in part required for growth in Swiss 3T3 cells.