Identification of autophosphorylation sites in c-Yes purified from rat liver plasma membranes.
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 LIEDNEYTAR, 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-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. 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-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.
Ariki
M
M
Department of Biochemistry, Hiroshima University School of Medicine.
Tanabe
O
O
Usui
H
H
Hayashi
H
H
Inoue
R
R
Nishito
Y
Y
Kagamiyama
H
H
Takeda
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics; D004586:Electrophoresis; D015972:Gene Expression Regulation, Neoplastic; D008099:Liver / Q000737:chemistry* / Q000166:cytology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051575:Proto-Oncogene Proteins c-yes; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases
D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D051575:Proto-Oncogene Proteins c-yes; C496639:Yes1 protein, mouse; D019061:src-Family Kinases