Identification of two epidermal growth factor-sensitive tyrosine phosphorylation sites of phospholipase C-gamma in intact HSC-1 cells

J Biol Chem. 1990 Mar 5;265(7):3944-8.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Brain / enzymology
  • Cattle
  • Cell Line
  • Epidermal Growth Factor / pharmacology*
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Phosphorylation
  • Phosphotyrosine
  • Sequence Homology, Nucleic Acid
  • Type C Phospholipases / isolation & purification
  • Type C Phospholipases / metabolism*
  • Tyrosine* / analogs & derivatives
  • Tyrosine* / analysis

Substances

  • Isoenzymes
  • Phosphotyrosine
  • Tyrosine
  • Epidermal Growth Factor
  • Type C Phospholipases