Regulation and Interaction of pp90rsk Isoforms with Mitogen-activated Protein Kinases*

Each of the three known mammalian 90-kDa S6 kinase (pp90rsk) isoforms (RSK1, RSK2, and RSK3) was expressed in transfected cells and further characterized. The kinase activity (immunocomplex toward S6 peptide) of each isoform was activated by in vivo growth factor (epidermal growth factor (EGF)) stimulation; RSK1 was more responsive (10-15-fold) versus RSK2 and RSK3 (2-4-fold). Pretreatment with PD98059 (MEK1 inhibitor) partially (80%) blocked EGF-mediated ERK1 activation and had similar effects on EGF stimulation of each ribosomal S6 kinase (RSK). Cotransfection with dominant-negative MEK1 inhibited activation of each RSK; furthermore, the kinase activity of RSK1, RSK2, and RSK3 was markedly increased by cotransfection with constitutively active MEK1. A specific association between mitogen-activated protein kinases (MAPKs) (ERK1 and ERK2) and RSK isoforms was tested by MAPK immunoblotting after immunoprecipitation of RSKs. ERK1 and ERK2 were present in RSK3 (and to a lesser extent, RSK2) immunoprecipitates, but were absent in RSK1 immunoprecipitates. Both dephosphorylated (from quiescent cells) and phosphorylated (from stimulated cells) MAPKs were associated with RSK2 and RSK3. Deletion mutants of RSK3 were characterized: the C terminus (33 residues) was shown to be required for association with MAPKs. The kinase activity of RSK1 or RSK2 was enhanced by in vitro incubation with ERK1. In contrast, RSK3 activity was not affected by exposure to ERK1. Furthermore, MAPKs in RSK3 immunoprecipitates were phosphorylated by purified MEK1; however, RSK3 kinase activity was unaffected. We conclude that 1) the MEK1-MAPK signaling pathway is both necessary and sufficient for in vivo growth factor-mediated activation of all three RSK isoforms; 2) RSK isoforms differ with respect to growth factor responsiveness and their physical association with MAPK; and 3) formation of the MAPK·RSK complex is mediated by the RSK C terminus.

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