Regulation of an epitope-tagged recombinant Rsk-1 S6 kinase by phorbol ester and erk/MAP kinase.

Phorbol ester tumor promoters (TPA) activate the endogenous erk/MAP kinases and Rsk S6 kinases but not the p70S6 kinase in COS cells. DNA sequences encoding the rat Rsk-1 S6 kinase (homologous to Xenopus rsk alpha), modified by insertion of a peptide epitope at the polypeptide aminoterminus, were expressed transiently in COS cells. TPA stimulates the 40S and peptide kinase activity of the recombinant epitope-tagged Rsk-1, as well as the extent of Rsk-1 autophosphorylation in vitro (32P-Ser >> 32P-Thr). Indications that the conformation of the recombinant Rsk-1 polypeptide is substantially changed after activation by TPA in situ include a retarded mobility of the Rsk-1 polypeptide on SDS-PAGE and the appearance of new 32P-peptides during autophosphorylation in vitro. All these features of the TPA-activated Rsk-1 S6 kinase are abolished by dephosphorylation of the kinase in vitro with Ser/Thr phosphatase-2A. TPA increases 32P incorporation into recombinant Rsk-1 by 2-3-fold (32P-Ser >> 32P-Thr). Peptide mapping exhibits a single major 32P-peptide in Rsk-1 isolated from unstimulated cells and 10-12 additional 32P peptides after TPA treatment in situ. Phosphorylation of basal or phosphatase-2A-treated recombinant Rsk-1 in vitro with erk2/MAP kinase increases Rsk-1 40S kinase, peptide kinase, and autophosphorylating activity, retards migration of Rsk-1 polypeptides on SDS-PAGE, and generates new sites of Rsk-1 autophosphorylation in vitro. By contrast, TPA-activated Rsk-1 is not altered in these properties by autophosphorylation in vitro. By contrast, TPA-activated Rsk-1 is not altered in these properties by phosphorylation in vitro with erk2/MAP kinase. Activation of Rsk-1 in situ with TPA diminishes by over 90% the extent of Rsk-1 phosphorylation achieved in vitro by erk2/MAP kinase, as compared to the parallel phosphorylation of a phosphatase-2A-treated Rsk-1; basal Rsk-1 is intermediate. Peptide maps of phosphatase-2A-treated Rsk-1 after phosphorylation in vitro with erk2/MAP kinase exhibit 32P-peptides that comigrate with nearly all of the 32P-peptides present in TPA-activated-32P Rsk-1 labeled in situ, plus several 32P-peptides characteristic of Rsk-1 autophosphorylation in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

[1]  J. Avruch,et al.  Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. , 1992, Science.

[2]  J. Bos,et al.  Involvement of p21ras in activation of extracellular signal-regulated kinase 2 , 1992, Nature.

[3]  J. Blenis,et al.  ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK , 1992, Cell.

[4]  J. Maller,et al.  A purified S6 kinase kinase from Xenopus eggs activates S6 kinase II and autophosphorylates on serine, threonine, and tyrosine residues. , 1992, The Journal of biological chemistry.

[5]  J. Avruch,et al.  An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase. , 1992, The Journal of biological chemistry.

[6]  J. Woodgett,et al.  Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini , 1991, Molecular and cellular biology.

[7]  James R. Woodgett,et al.  Phosphorylation of c-jun mediated by MAP kinases , 1991, Nature.

[8]  T. Curran,et al.  Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. , 1991, The Journal of biological chemistry.

[9]  P. Cohen,et al.  Purification and characterisation of the insulin-stimulated protein kinase from rabbit skeletal muscle; close similarity to S6 kinase II. , 1991, European journal of biochemistry.

[10]  J. Blenis,et al.  Mitogen-activated Swiss mouse 3T3 RSK kinases I and II are related to pp44mpk from sea star oocytes and participate in the regulation of pp90rsk activity. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[11]  R. Erikson,et al.  Structure, expression, and regulation of protein kinases involved in the phosphorylation of ribosomal protein S6. , 1991, The Journal of biological chemistry.

[12]  J. Blenis,et al.  Coordinate regulation of pp90rsk and a distinct protein-serine/threonine kinase activity that phosphorylates recombinant pp90rsk in vitro , 1991, Molecular and cellular biology.

[13]  J. Maller,et al.  Purification and characterization of ribosomal protein S6 kinase I from Xenopus eggs. , 1991, The Journal of biological chemistry.

[14]  J. Avruch,et al.  Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Siegmann,et al.  Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Avruch,et al.  Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[17]  E. Krebs,et al.  Evidence for an epidermal growth factor-stimulated protein kinase cascade in Swiss 3T3 cells. Activation of serine peptide kinase activity by myelin basic protein kinases in vitro. , 1990, The Journal of biological chemistry.

[18]  R. Erikson,et al.  Coinfection of insect cells with recombinant baculovirus expressing pp60v-src results in the activation of a serine-specific protein kinase pp90rsk. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Dixon,et al.  Cloning and expression of a protein-tyrosine-phosphatase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Alcorta,et al.  Sequence and expression of chicken and mouse rsk: homologs of Xenopus laevis ribosomal S6 kinase , 1989, Molecular and cellular biology.

[21]  J. Maller,et al.  In vivo phosphorylation and activation of ribosomal protein S6 kinases during Xenopus oocyte maturation. , 1989, The Journal of biological chemistry.

[22]  J. Avruch,et al.  Purification of a hepatic S6 kinase from cycloheximide-treated Rats. , 1989, The Journal of biological chemistry.

[23]  V. Pathak,et al.  The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs , 1989, Molecular and cellular biology.

[24]  J. Maller,et al.  Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II , 1988, Nature.

[25]  J. Blenis,et al.  A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Wigler,et al.  Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method , 1988, Molecular and cellular biology.

[27]  J. Blenis,et al.  Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts , 1987, Molecular and cellular biology.

[28]  J. Maller,et al.  Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6. , 1986, The Journal of biological chemistry.

[29]  Andrew R. Cherenson,et al.  The structure of an antigenic determinant in a protein , 1984, Cell.

[30]  R. Lerner,et al.  Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[31]  H. Towbin,et al.  Criteria for establishment of the biological significance of ribosomal protein phosphorylation. , 1982, Current topics in cellular regulation.