Characterization of an activated ribosomal S6 kinase variant from maturing sea star oocytes: Association with phosphatase 2A and substrate specificity
✍ Scribed by Lorin A. Charlton; Mohamed Sayed; Ian Clark-Lewis; Ruedi Aebersold; Steven L. Pelech
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 386 KB
- Volume
- 75
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Two ribosomal protein S6 kinases (i.e., pp52 S6K and pp70 S6K ) of the p70 S6 kinase family were markedly activated during meiotic maturation of Pisaster ochraceus sea star oocytes. A rapid protocol was developed for the purification from the oocyte cytosol of pp52 S6K by ϳ50,000-fold with a specific enzyme activity of 1.6 µmol per min per mg. The purified enzyme apparently featured the N-and C-terminal regions of pp70 S6K as it immunoreacted with antibodies directed to peptides patterned after these amino acid sequences in mammalian pp70 S6K . pp52 S6K was inhibited by fluoride (IC 50 ϳ60 mM), but was relatively insensitive to -glycerolphosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions such as Mn 2ϩ , Zn 2ϩ , and Ca 2ϩ . The consensus sequence for substrate phosphorylation was determined to be RXXSXR, which was partially distinct from mammalian p70 S6K in its requirement for an amino-terminal arginine. Phosphorylation of ribosomal protein S6 by p52 S6K occurred exclusively on serine on at least five tryptic peptides. Inhibition of sea star p52 S6K phosphotransferase activity after treatment with protein serine/threonine phosphatases confirmed that p52 S6K was still regulated by phosphorylation. The sea star S6 kinase was purified to near homogeneity with the regulatory and catalytic subunits of protein-serine phosphatase 2A and the heat shock protein 60. The association of an S6 kinase with phosphatase 2A was confirmed by coimmunoprecipitation of S6 kinase activity with phosphatase 2A-specific antibodies. The purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosphorylation of the S6 protein and other targets.