Identification of two epidermal growth factor-sensitive tyrosine phosphorylation sites of phospholipase C-gamma in intact HSC-1 cells.
The 145-kDa phospholipase C isozyme, PLC-gamma, is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. We now demonstrate that EGF treatment of HSC-1 cells, a human squamous cell carcinoma-derived cell line that expresses high levels of the EGF receptor, rapidly induces tyrosine phosphorylation of two-thirds of the total cellular PLC-gamma pool. A two-step immunoaffinity protocol was used for large-scale isolation of phosphorylated PLC-gamma from the cytosol of EGF-treated HSC-1 cells. Phosphorylated PLC-gamma was digested with trypsin, then phosphotyrosine-containing peptides were purified by phosphotyrosine affinity chromatography and reverse-phase high performance liquid chromatography. The two major phosphotyrosine-containing tryptic peptides were sequenced. Comparison of the sequence data with the bovine brain PLC-gamma amino acid sequence indicated that the major, EGF-sensitive tyrosine phosphorylation sites of human PLC-gamma correspond to the bovine brain PLC-gamma tyrosine residues 771 and 1254. The former residue is adjacent to regions of PLC-gamma that contain high homology to the non-catalytic, amino-terminal region of the src tyrosine kinase. The latter residue lies near the carboxyl terminus of the PLC-gamma molecule. The accompanying manuscript (Kim J.W., Sim, S.S., Kim, U-H., Nishibe, S., Wahl, M. I., Carpenter, G., and Rhe, S. G. (1990) J. Biol. Chem. 265, 3940-3943) identifies these same 2 residues plus 2 additional tyrosine phosphorylation sites through large-scale in vitro phosphorylation of purified bovine brain PLC-gamma by the EGF receptor.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
M I
MI
Wahl
S
S
Nishibe
J W
JW
Kim
H
H
Kim
S G
SG
Rhee
G
G
Carpenter
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D019000:Phosphotyrosine; D012689:Sequence Homology, Nucleic Acid; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D007527:Isoenzymes; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D010738:Type C Phospholipases