Characterization of an activated ribosomal S6 kinase variant from maturing sea 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 Pisaster ochraceus sea star oocytes. A rapid protocol was developed for the purification from the oocyte cytosol of pp52(S6K) by approximately 50,000-fold with a specific enzyme activity of 1.6 micromol 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) approximately 60 mM), but was relatively insensitive to beta-glycerolphosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions such as Mn(2+), Zn(2+), and Ca(2+). 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.
Charlton
L A
LA
Department of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Sayed
M
M
Clark-Lewis
I
I
Aebersold
R
R
Pelech
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D015151:Immunoblotting; D007477:Ions; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D054648:Protein Phosphatase 2; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism; D017421:Sequence Analysis; D016622:Silver Staining; D013215:Starfish / Q000196:embryology / Q000201:enzymology; D013379:Substrate Specificity
D007477:Ions; D012269:Ribosomal Proteins; D019893:Ribosomal Protein S6 Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2