Purification and Characterization of a Src-related p57 Protein-tyrosine Kinase from Xenopus Oocytes

Abstract

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 proteintyrosine 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.

Are Src family kinases involved in cell cycle resumption in rat eggs?

“…Fertilization is known to stimulate the SFKs in eggs of some vertebrates such as Xenopus and zebrafish (Sato et al 1996, Wu & Kinsey 2001. Furthermore, SFKs inhibitors interrupt sperm-egg fusion in frogs (Sato et al 1999(Sato et al , 2000a.…”

“…In vertebrate eggs, the mechanism leading to IP 3 production and to Ca 2þ rise during fertilization is less established. The SFKs known to be expressed in vertebrate eggs are: c-Src kinase, c-Yes kinase and a 57 kDa Src-related kinase in Xenopus laevis (Schartl & Barnekow 1984, Steele et al 1989, Sato et al 1996; Fyn kinase in Xenopus, zebrafish, rat and mouse (Kinsey 1996, Talmor et al 1998, Wu & Kinsey 2001, Sette et al 2002.…”

Biochemical Characterization of the Arabidopsis Protein Kinase SOS2 That Functions in Salt Tolerance

“…Regulation of protein kinases is achieved through many different mechanisms, including protein phosphorylation by other kinase(s) (Elion, 1998), autophosphorylation (Cooper andMacAuley, 1988;Sato et al, 1996), or control by regulatory domains or subunits. A key feature for regulation in many protein kinases is thought to be the phosphorylation of one or more residues within the activation loop of the catalytic subunit (Vertommen et al, 2000;McCartney and Schmidt, 2001).…”

SH2 domain-mediated activation of an SRC family kinase is not required to initiate Ca2+ release at fertilization in mouse eggs

“…In mammals (Miyazaki et al 1992(Miyazaki et al , 1993, as well as other species in the vertebrate evolutionary line (see Runft et al 2002), the Ca 2þ is released from the egg's endoplasmic reticulum by inositol trisphosphate (IP 3 ), but the mechanisms leading to IP 3 production are incompletely understood. SRC family kinases (SFKs) function in this signalling pathway in echinoderms (Jaffe et al 2001, ascidians (Runft & Jaffe 2000), fish (Wu & Kinsey 2002, Kinsey et al 2003, and frogs (Sato et al 1996(Sato et al , 2000, but whether the same is true in mammals is uncertain.…”