Purification and characterization of a rat liver membrane tyrosine-protein kinase, the possible protooncogene c-yes product, p60c-yes.
A tyrosine-protein kinase was purified more than 270-fold from the rat liver plasma membrane fraction by successive column chromatographies on Sephacryl S-300, wheat germ agglutinin-agarose, casein-Sepharose, and hydroxylapatite, followed by isoelectrofocusing electrophoresis. The enzyme with pI of 6.2 was a 60-kDa single polypeptide which represented 42% of total protein. The enzyme reacted quantitatively with a monoclonal antibody to the amino-terminal sequence (Cys-3 to Ser-66) specific to the human c-yes protein, but not with antibodies to the specific amino-terminal sequences of the c-src, fyn, and lck proteins. The purified enzyme contained almost no phosphotyrosine residue but was autophosphorylated with Mg.ATP exclusively at tyrosine residues with concomitant increase in the kinase activity. The rates of autophosphorylation of the enzyme and phosphorylation of tyrosine-glutamate (1:4) copolymers, catalyzed by the enzyme were proportional to the square of enzyme concentration, suggesting that p60c-yes undergoes autophosphorylation through intermolecular catalysis, resulting in stimulation of the enzyme activity. Although the enzyme reaction showed an essential requirement for Mg2+ or Mn2+ with optimal concentrations of 20 and 3 mM, respectively, autophosphorylation significantly activated the enzyme only in the presence of Mg2+. Autophosphorylation of the enzyme reduced the Km for tyrosine-glutamate copolymers and tubulin, but not for ATP, and increased the Vmax of copolymer and tubulin phosphorylation.
Azuma
K
K
Department of Biochemistry, Hiroshima University School of Medicine, Japan.
Ariki
M
M
Miyauchi
T
T
Usui
H
H
Takeda
M
M
Semba
K
K
Matsuzawa
Y
Y
Yamamoto
T
T
Toyoshima
K
K
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification*; D051575:Proto-Oncogene Proteins c-yes; D051381:Rats; D019061:src-Family Kinases
D011518:Proto-Oncogene Proteins; D008345:Manganese; D011505:Protein-Tyrosine Kinases; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases; D008274:Magnesium