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 ki nases and Rsk S6 kinases but not the p70 S6 kinase in COS cells. DNA s equences encoding the rat Rsk-1 S6 kinase (homologous to Xenopus rskal pha), modified by insertion of a peptide epitope at the polypeptide am inoterminus, were expressed transiently in COS cells. TPA stimulates t he 40S and peptide kinase activity of the recombinant epitope-tagged R sk-1, as well as the extent of Rsk-1 autophosphorylation in vitro (P-3 2-Ser >> P-32-Thr). Indications that the conformation of the recombina nt Rsk-1 polypeptide is substantially changed after activation by TPA in situ include a retarded mobility of the Rsk-1 polypeptide on SDS-PA GE 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 pho sphatase-2A. TPA increases P-32 incorporation into recombinant Rsk-1 b y 2-3-fold (P-32-Ser >> P-32-Thr). Peptide mapping exhibits a single m ajor 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 erk 2/MAP kinase increases Rsk-1 40S kinase, peptide kinase, and autophosp horylating activity, retards migration of Rsk-1 polypeptides on SDS-PA GE, and generates new sites of Rsk-1 autophosphorylation in vitro. By contrast, TPA-activated Rsk-1 is not altered in these properties by ph osphorylation in vitro with erk2/MAP kinase. Activation of Rsk-1 in si tu with TPA diminishes by over 90% the extent of Rsk-1 phosphorylation achieved in vitro by erk2/MAP kinase, as compared to the parallel pho sphorylation of a phosphatase-2A-treated Rsk-1; basal Rsk-1 is interme diate. Peptide maps of phosphatase-2A-treated Rsk-1 after phosphorylat ion 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 Rs k-1 labeled in situ, plus several P-32-peptides characteristic of Rsk- 1 autophosphorylation in vitro. If Rsk-1 is heat inactivated to preven t autophosphorylation during treatment with erk2/MAP kinase in vitro, a greatly simplified Rsk-1 peptide map (P-32-Thr > P-32-Ser) is obtain ed that lacks not only the characteristic sites of Rsk-1 in vitro auto phosphorylation but many of the P-32-peptides that comigrate with P-32 -Rsk-1 peptides labeled in situ during TPA activation. Thus, erk/MAP k inase or enzymes with very similar specificity are likely the sole ups tream 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.