A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation.
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.
Chung
Sang Hee
SH
Department of Life Science, College of Natural Science, Daejin University, Pocheon 487-711, Korea.
Kim
Sung Kuk
SK
Kim
Jung Kuk
JK
Yang
Yong Ryoul
YR
Suh
Pann Ghill
PG
Chang
Jong Soo
JS
D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006868:Hydrolysis / Q000187:drug effects; D050505:Mutant Proteins / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics*; D051381:Rats
D050505:Mutant Proteins; D010716:Phosphatidylinositols; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D051966:Phospholipase C gamma