Identification of autophosphorylation sites in c-Yes purified from rat liver plasma membranes

Masahiro Ariki, Osamu Tanabe, Hirofumi Usui, Hideyuki Hayashi, Rintaro Inoue, Yasumasa Nishito, Hiroyuki Kagamiyama, Masao Takeda

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEY?TAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-18 through Lys-46, Y?TPENPTEPVNTSAGH?YGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-18 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.

Original languageEnglish
Pages (from-to)104-111
Number of pages8
JournalJournal of biochemistry
Volume121
Issue number1
DOIs
Publication statusPublished - 1997 Jan 1

Keywords

  • Autophosphorylation sites
  • N-terminal sequence
  • Purification
  • Rat liver plasma membrane
  • c-Yes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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