p90 ribosomal S6 kinase and p70 ribosomal S6 kinase link phosphorylation of the eukaryotic chaperonin containing TCP-1 to growth factor, insulin, and nutrient signaling

Abstract

Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 ribosomal S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K). RSK phosphorylates the β subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein kinase (MAPK) pathway. CCTβ Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTβ phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTβ Ser-260 phosphorylation in response to multiple agonists in various mammalian cells.Wealso show that RNA interference-mediated knockdown of endogenous CCTβ causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTβ wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTβ regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.