Abstract
S6 kinases I and II have been purified previously from Xenopus eggs and shown to be activated by phosphorylation on serine and threonine residues. An S6 kinase clone, closely related to S6 kinase II, was subsequently identified and the protein product was expressed in a baculovirus system. Using this protein, termed "rsk" for Ribosomal Protein S6 Kinase, as a substrate, we have purified to homogeneity from unfertilized Xenopus eggs a 41-kDa serine/threonine kinase termed rsk kinase. Both microtubule-associated protein-2 and myelin basic protein are good substrates for rsk kinase, whereas alpha-casein, histone H1, protamine, and phosvitin are not. rsk kinase is inhibited by low concentrations of heparin as well as by beta-glycerophosphate and calcium. Activation of rsk kinase during Xenopus oocyte maturation is correlated with phosphorylation on threonine and tyrosine residues. However, in vitro, rsk kinase undergoes autophosphorylation on serine, threonine, and tyrosine residues, identifying it as a "dual specificity" enzyme. Purified rsk kinase can be inactivated in vitro by either a 37-kDa T-cell protein-tyrosine phosphatase or the serine/threonine protein phosphatase 2A. Phosphatase-treated S6KII can be reactivated by rsk kinase, and S6 kinase activity in resting oocyte extracts increases significantly when purified rsk kinase is added. The availability of purified rsk kinase will enhance study of the signal transduction pathway(s) regulating phosphorylation of ribosomal protein S6 in Xenopus oocytes.
Highlights
The substrate specificity of purified rsk kinase was examined (Table 111). Both MAP-2 andmyelin basic protein were phosphorylated at 30-40 times the rate of rsk, but this difference may be due in part to thehigher concentration of MAP-2 and myelin basic protein in the assays. a-Casein and histone H 1 were phosphorylated at a lower rate thanrsk, and protamine, phosvitin, and 40 S ribosomal subunits were poor substrates
The effects of the serine/threonine protein phosphatases1 and 2A, the protein tyrosine phosphatase CD45, andthe truncated 37-kDa T-cell phosphatase on rsk kinase activity were examined. rsk kinase activity was not reduced in the presence of 10 units/ml of phosphatase 1, whereas the same concentration of phosphatase 1 decreased S6 kinase I1 activity by more than 50%
5% of rsk kinase activity remained after 60 min, whereas almost 80% of the activity remained when rsk kinase was treated with phosphatase 2A inactivated with NaF
Summary
Rsk Kinase Purification-Unfertilized Xenopus eggs arrested at metaphase of meiosis I1 exhibit maximal S6 kinase activity [8]and were used as asource of rsk kinase. The activity eluted as avery broad peak, well after the protein markers and the p-nitrophenyl phosphate (Fig. 2). The sample was chromatographed on a phenyl-TSK column (Fig. 3) with a gradient of increasing ethylene glycol and decreasing NaCl(l7). A silver-stained polyacrylamide gel of the pooled peak fractions (Fig. 4) shows thattheTSK G3000SWcolumn resolved most contaminating proteinsfrom the rsk kinase, and only a 41-42-kDa doublet was observed following chromatography on the phenyl column. This doublet probably represents two isoforms of the same protein since. Rsk kinase purified without the addition of p-nitrophenyl phosphate to theDEAE fractions shows an increased proportion of protein in the faster migrating form.
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