Exp Mol Med.  2010 Mar;42(3):216-222. 10.3858/emm.2010.42.3.023.

A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

• Chung SH
• Kim SK
• Kim JK
• Yang YR
• Suh PG
• Chang JS

• KMID: 946654
• DOI: http://doi.org/10.3858/emm.2010.42.3.023

Abstract

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

Keyword

phosphatidylinositol 4,5-bisphosphate; phospholipase Cgamma; protein-tyrosine kinases; src homology domains

MeSH Terms

Amino Acid Substitution/drug effects/*genetics
Animals
COS Cells
Cercopithecus aethiops
Enzyme Activation/drug effects
Epidermal Growth Factor/*pharmacology
Hydrolysis/drug effects
Mutant Proteins/metabolism
Phosphatidylinositols/*metabolism
Phospholipase C gamma/*genetics/metabolism
Phosphorylation/drug effects
Phosphotyrosine/*metabolism
Point Mutation/*genetics
Rats