Purification of recombinant SH2/SH3 proteins of phospholipase C-gamma 1 and -gamma 2 and their inhibitory effect on PIP2-hydrolysis induced by both types of phospholipase C-gamma.
In order to examine physiological function of the SH2/SH3 region of phospholipase C-gamma (Z region), we independently expressed cDNA fragments corresponding to the SH2/SH3 region of PLC-gamma 1 and PLC-gamma 2 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-gamma 1 and designated as rP45Z) and 38 kDa (derived from PLC-gamma 2 and designated as rP38Z), which was consistent with that as expected from inserted cDNA. We determined the effect of purified rP45Z or rP38Z on PIP2-hydrolyzing activity of either PLC-gamma 1 or PLC-gamma 2 and found that these proteins strongly suppressed the rate of PLC-dependent PIP2-hydrolysis. Furthermore, both rP45Z and rP38Z were phosphorylated at tyrosine residue by epidermal growth factor receptors and their inhibitory effect on PIP2-hydrolysis was significantly decreased by this phosphorylation. These results indicate that the Z region might be involved in autoregulation of PLC-gamma as intrinsic negative regulator.
Homma
Y
Y
Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Japan.
Emori
Y
Y
Takenawa
T
T
D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D066246:ErbB Receptors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D004247:DNA; D066246:ErbB Receptors; D010738:Type C Phospholipases