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

• Published in: Biochemistry (Easton) Vol. 32; no. 30; pp. 7727 - 7738
• Main Authors: Grove, J. Russell, Price, Daniel J, Banerjee, Papia, Balasubramanyam, Ashok, Ahmad, Mir F, Avruch, Joseph
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 03.08.1993
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Cell physiology; Cells, Cultured; DNA; Enzyme Activation; Epitopes; Fundamental and applied biological sciences. Psychology; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Molecular and cellular biology; Molecular Sequence Data; Phosphorylation; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Protein-Tyrosine Kinases - metabolism; Rats; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Responses to growth factors, tumor promotors, other factors; Ribosomal Protein S6 Kinases; Tetradecanoylphorbol Acetate - pharmacology; Xenopus; Autophosphorylation; Phosphorylation; Heterologous system; Rat; Nucleotide sequence; Enzyme; Rodentia; Monkey; Activation; In vitro; Signal transduction; Vertebrata; Mammalia; Cell line; Complementary DNA; Protein kinase; Primates
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00081a018

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.