Characterization of an activated ribosomal S6 kinase variant from maturingsea star oocytes: Association with phosphatase 2A and substrate specificity

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 Pisa ster ochraceus sea star oocytes. A rapid protocol was developed for the pur ification from the oocyte cytosol of pp52(S6K) by similar to 50,000-fold wi th a specific enzyme activity of 1.6 mu mol per min per mg. The purified en zyme apparently featured the N- and C-terminal regions of pp70(S6K) as it i mmunoreacted with antibodies directed to peptides patterned after these ami no acid sequences in mammalian pp70(S6K) pp52(S6K) was inhibited by fluorid e (IC50 similar to 60 mM), but was relatively insensitive to beta-glycerolp hosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions suc h as Mn2+, Zn2+, and Ca2+. The consensus sequence for substrate phosphoryla tion was determined to be RXXSXR, which was partially distinct from mammali an p70(S6K) in its requirement for an amino-terminal arginine. Phosphorylat ion of ribosomal protein S6 by p52(S6K) occurred exclusively on serine on a t least five tryptic peptides. Inhibition of sea star p52(S6K) phosphotrans ferase activity after treatment with protein serine/threonine phosphatases confirmed that p52(S6K) was Still regulated by phosphorylation. The sea sta r S6 kinase was purified to near homogeneity with the regulatory and cataly tic subunits of protein-serine phosphatase 2A and the heat shock protein 60 . The association of an S6 kinase with phosphatase 2A was confirmed by coim munoprecipitation of S6 kinase activity with phosphatase 2A-specific antibo dies. The purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosp horylation of the S6 protein and other targets. J. Cell. Biochem. 75:310-32 6, 1999. (C) 1999 Wiley-Liss, Inc.