Purification of recombinant [formula omitted] proteins of phospholipase C-γ1 and -γ2 and their inhibitory effect on PIP 2-hydrolysis induced by both types of phospholipase C-γ

Abstract

In order to examine physiological function of the SH2 SH3 region of phospholipase C-γ (Z region), we independently expressed cDNA fragments corresponding to the SH2 SH3 region of PLC-γ1 and PLC-γ2 in Escherichia coli. Although these recombinant proteins were recovered in particulate fractions by centrifugation of cell extracts, they were successfully solubilized by guanidium hydrochloride and then purified to homogeneity by heparin column chromatography. The molecular mass of the proteins was 45 kDa (derived from PLC-γ1 and designated as rP45Z) and 38 kDa (derived from PLC-γ2 and designated as rP38Z), which was consistent with that as expected from inserted cDNA. We determined the effect of purified rP45Z or rP38Z on PIP 2-hydrolyzing activity of either PLC-γ1 or PLC-γ2 and found that these proteins strongly suppressed the rate of PLC-dependent PIP 2-hydrolysis. Furthermore, both rP45Z and rP38Z were phosphorylated at tyrosine residue by epidermal growth factor receptors and their inhibitory effect on PIP 2-hydrolysis was significantly decreased by this phosphorylation. These results indicate that the Z region might be involved in autoregulation of PLC-γ as intrinsic negative regulator.