Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6.
In Xenopus oocytes ribosomal protein S6 becomes phosphorylated on serine residues in response to hormones or growth factors and following microinjection of the tyrosine-specific protein kinases associated with Rous sarcoma virus or Abelson murine leukemia virus. To begin characterization of the enzymes responsible for S6 phosphorylation in this system, we have undertaken the purification of S6 protein kinases from unfertilized Xenopus eggs. DEAE-Sephacel chromatography of crude extracts revealed two peaks of S6 kinase activity, and the peak eluting at 160 mM NaCl was chosen for further purification. Successive chromatography on Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose resulted in purification of the enzyme to a single protein migrating at Mr = 92,000 on polyacrylamide gels. The final preparation was purified about 500-fold from the DEAE-Sephacel peak with a recovery of 10%. Apparent Km values of the enzyme for ATP and 40 S subunits were 28 and 5 microM, respectively, and the specific activity with 330 microM ATP and 5.6 microM 40 S subunits was 300 nmol/min/mg. The enzyme was inhibited by beta-glycerophosphate, sodium fluoride, potassium phosphate, ADP, heparin, quercetin, and spermine. The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D009865:Oocytes / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*; D014982:Xenopus laevis
D009842:Oligopeptides; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D008345:Manganese; C037642:kemptide; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D008274:Magnesium