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 p70 S6 kinase in COS cells. DNA sequences encoding the rat Rsk-1 S6 kinase (homologous to Xenopus rskalpha), 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 (P-32-Ser >> P-32-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 P-32-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 P-32 incorporation into recombinant Rsk-1 by 2-3-fold (P-32-Ser >> P-32-Thr). Peptide mapping exhibits a single major P-32-peptide in Rsk-1 isolated from unstimulated cells and 10-12 additional P-32 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 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 P-32-peptides that comigrate with nearly all of the P-32-peptides present in TPA-activated-P-32 Rsk-1 labeled in situ, plus several P-32-peptides characteristic of Rsk-1 autophosphorylation in vitro. If Rsk-1 is heat inactivated to prevent autophosphorylation during treatment with erk2/MAP kinase in vitro, a greatly simplified Rsk-1 peptide map (P-32-Thr > P-32-Ser) is obtained that lacks not only the characteristic sites of Rsk-1 in vitro autophosphorylation but many of the P-32-peptides that comigrate with P-32-Rsk-1 peptides labeled in situ during TPA activation. Thus, erk/MAP kinase or enzymes with very similar specificity are likely the sole upstream activator mediating TPA stimulation of Rsk-1 kinase in COS cells and act together with Rsk-1 autophosphorylation to achieve the final Rsk-1 phosphorylation state observed in situ.