Selectivity of phospholipase C phosphorylation by the epidermal growth factor receptor, the insulin receptor, and their cytoplasmic domains.
Phosphatidylinositol-specific phospholipase C isozyme gamma (PLC-gamma, Mr 145,000) is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. PLC-beta-1, another PLC isozyme, is a poor substrate for the EGF receptor. We examined the relative phosphorylation of PLC-gamma and PLC-beta-1 by the 170-kDa native EGF receptor molecule, the 66-kDa cytoplasmic kinase domain of the EGF receptor (Arg647-Ala1186), the alpha 2 beta 2 native insulin receptor, and the 48-kDa cytoplasmic kinase domain of the insulin receptor beta subunit (Gly947-Ser1343). Similar to the intact EGF receptor, the cytoplasmic kinase domain of the EGF receptor preferentially phosphorylated PLC-gamma. High-performance liquid chromatographic comparison of tryptic phosphopeptides from PLC-gamma phosphorylated by both forms of the EGF receptor kinase indicated similar patterns of multiple tyrosine phosphorylations. These results imply that substrate selectivity, at least in terms of PLC isozymes, is independent of the extracellular ligand-binding and membrane anchor domains of the EGF receptor. In comparison, neither the intact insulin receptor nor the beta-chain kinase domain was able to phosphorylate PLC-gamma to a significant extent. Also, insulin failed to stimulate the phosphorylation of PLC-gamma in NIH 3T3/HIR cells, which overexpress the human insulin receptor. Thus PLC-gamma is not a phosphorylation substrate for the insulin receptor in vitro or in the intact cell.
Nishibe
S
S
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146.
Wahl
M I
MI
Wedegaertner
P B
PB
Kim
J W
JW
Rhee
S G
SG
Carpenter
G
G
Kim
J J
JJ
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D014357:Trypsin; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D010748:Phosphopeptides; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D010738:Type C Phospholipases; D014357:Trypsin