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Regulation of an epitope-tagged recombinant Rsk-1 S6 kinase by phorbol ester and erk/MAP kinase
J R Grove1, D J Price, P Banerjee
1Diabetes Unit, Massachusetts General Hospital, Boston.
Abstract:
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)
Insights
Phorbol ester tumor promoters (TPA) activate Rsk-1 S6 kinase, increasing its activity and altering its phosphorylation. This TPA-induced activation is reversed by phosphatase treatment, highlighting its role in kinase regulation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Kinase Biology
Background:
- Phorbol ester tumor promoters (TPA) are known activators of cellular signaling pathways.
- Rsk S6 kinases play crucial roles in cell growth and proliferation.
- Understanding the regulation of Rsk kinases is vital for deciphering cellular responses to stimuli.
Purpose of the Study:
- To investigate the activation mechanism of Rsk-1 S6 kinase by TPA in COS cells.
- To characterize the conformational and activity changes in Rsk-1 upon TPA stimulation.
- To elucidate the interplay between TPA-induced activation and MAP kinase phosphorylation.
Main Methods:
- Transient expression of epitope-tagged rat Rsk-1 in COS cells.
- Treatment with TPA and subsequent analysis of kinase activity, autophosphorylation, and SDS-PAGE mobility.
- In vitro dephosphorylation using Ser/Thr phosphatase-2A.
- In vitro phosphorylation using erk2/MAP kinase and peptide mapping.
Main Results:
- TPA activates Rsk-1 S6 kinase, increasing its 40S and peptide kinase activities and autophosphorylation.
- TPA-induced activation causes conformational changes in Rsk-1, evidenced by altered SDS-PAGE mobility and new phosphorylation sites.
- TPA activation diminishes subsequent in vitro phosphorylation by erk2/MAP kinase, suggesting a feedback mechanism.
Conclusions:
- TPA triggers a significant activation and conformational change in Rsk-1 S6 kinase.
- Phosphorylation by erk2/MAP kinase contributes to Rsk-1 activation, but TPA-induced activation involves additional regulatory steps.
- Dephosphorylation by Ser/Thr phosphatase-2A reverses TPA-mediated activation and phosphorylation changes.