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 beta subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein kinase (MAPK) pathway. CCTbeta 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 CCTbeta phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTbeta Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTbeta causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTbeta wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTbeta regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.
Highlights
Distinct families of Ser/Thr kinases that regulate diverse cellular processes
As a first step in addressing this potential link between growth factor signaling and the biological processes regulated by chaperonin containing TCP-1 (CCT), we show that ribosomal S6 kinase (RSK) and S6 kinase (S6K) phosphorylate the CCT subunit at Ser-260
phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) Pathway—After confirming that RSK phosphorylates CCT on Ser-260 in phorbol myristate acetate (PMA)- or epidermal growth factor (EGF)-treated HEK293E cells, we examined whether CCT is phosphorylated by S6K
Summary
Materials—Anti-CCT␣, anti-CCT, anti-CCT, and antiactin antibodies were obtained from Santa Cruz Biotechnology. Our findings indicate that RSK phosphorylates CCT in vitro and is the major kinase responsible for CCT phosphorylation in EGF- or PMA-stimulated HEK293E cells. CCT Ser-260 Is the Phosphorylation Site Recognized by ␣PAS—To identify potential RSK phosphorylation sites, the immunoprecipitated FLAG-CCT was digested in-gel with trypsin, chymotrypsin, or Lys-C and subjected to LC-MS/MS analysis equipped with both collision-induced dissociation and electron transfer dissociation. To further demonstrate that CCT is phosphorylated in a RSK- S260A, indicating that phospho-Ser-260 is recognized by ␣PAS dependent manner, we treated cells with several pharmacolog- (Fig. 3B). Activated HA-tagged RSK1 phosphorylated wildtype CCT protein, but did not phosphorylate the S260A protein, confirming that Ser-260 is the major site phosphorylated by RSK (Fig. 3C) These results indicate that RSK directly phosphorylates CCT Ser-260 in vitro and in intact cells. Overexpression of S6K1 resulted in enhanced Ser-260 phosphorylation, but not for CCT S260A (Fig. 4D)
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