Purification and characterization of a Src-related p57 protein-tyrosine kinase from Xenopus oocytes. Isolation of an inactive form of the enzyme and its activation and translocation upon fertilization.
In the previous study (Fukami, Y., Sato, K.-I., Ikeda, K., Kamisango, K., Koizumi, K., and Matsuno, T. (1993) J. Biol. Chem. 268, 1132-1140), we found that an antibody termed anti-pepY antibody causes a severalfold activation of bovine brain c-Src. The anti-pepY antibody was raised against a synthetic peptide corresponding to residues 410-428 of chicken c-Src, one of the most conserved regions among the Src family protein-tyrosine kinases. In this study, we have used this antibody as an in vitro activator and purified a c-Src-related protein-tyrosine kinase from the particulate fraction of Xenopus laevis oocytes. A synthetic peptide corresponding to residues 7-26 of fission yeast Cdc2 was used as substrate. Immunoreactivity toward the antibody was also monitored during the purification. The purified kinase displayed a single polypeptide of 57 kDa on SDS-gel electrophoresis and showed a specific activity of 2.37 and 20.1 nmol/min/mg protein in the absence and the presence of the anti-pepY antibody, respectively. The purified enzyme underwent autophosphorylation and phosphorylated actin and the Cdc2 peptide exclusively on tyrosine residues. Specific antibodies against c-Src, Fyn, c-Yes, c-Fgr, Lck, Lyn, Hck, and Blk proteins did not recognize the p57 Xenopus tyrosine kinase. The kinase activity of the Xenopus enzyme was not affected by oocyte maturation but was found to be elevated severalfold upon fertilization. Fertilization also caused a translocation of the activated enzyme from the particulate fraction to the cytosolic fraction. The activation and translocation was observed within 1 min after fertilization. These results suggest a possible involvement of the p57 Xenopus tyrosine kinase in the signal transduction of fertilization.
Sato
K
K
Laboratory of Molecular Biology, Biosignal Research Center and the Graduate School of Science and Technology, Kobe University, Nada, Kobe 657, Japan.
Aoto
M
M
Mori
K
K
Akasofu
S
S
Tokmakov
A A
AA
Sahara
S
S
Fukami
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004789:Enzyme Activation; D005260:Female; D005306:Fertilization; D007120:Immunochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism; D014982:Xenopus laevis / Q000196:embryology; D019061:src-Family Kinases / Q000302:isolation & purification* / Q000378:metabolism
D014443:Tyrosine; D019061:src-Family Kinases