The CDK inhibitor p27Kip1 plays a central role in controlling cell proliferation and cell-cycle exit. p27Kip1 protein levels oscillate during cell-cycle progression and are regulated by mitogen or anti-proliferative signaling. The abundance of the protein is frequently determined by post-transcriptional mechanisms including ubiquitin-mediated proteolysis and translational control. Here, we report that the cold-inducible RNA-binding protein (CIRP) selectively binds to the 5′ untranslated region of the p27Kip1 mRNA. CIRP is induced, modified and relocalized in response to various stress stimuli and can regulate cell survival and cell proliferation particularly during stress. Binding of CIRP to the 5′UTR of the p27Kip1 mRNA significantly enhanced reporter translation. In cells exposed to mild hypothermia, the induction of CIRP correlated with increased translation of a p27Kip1 5′UTR reporter and with the accumulation of p27Kip1 protein. shRNA-mediated CIRP knockdown could prevent the induction of translation. We found that p27Kip1 is central for the decreased proliferation at lower temperature, since p27Kip1 KO mouse embryonic fibroblasts (MEFs) hardly increased their doubling time in hypothermic conditions, whereas wild-type MEFs significantly delayed proliferation in response to cold stress. This suggests that the CIRP-dependent p27Kip1 upregulation during mild hypothermia contributes to the cold shock-induced inhibition of cell proliferation.
The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57 Kip2 is a member of the cip/Kip family of cKis and frequently inactivated by genomic mutations associated with human overgrowth disorders. There is increasing evidence for p57 to control cellular processes in addition to cell cycle and cDK regulation including transcription, apoptosis, migration or development. In order to obtain molecular insights to unknown functions of p57, we performed a protein interaction screen. We identified the transcription regulator four-and-a-half LIM-only protein 2 (FHL2) as a novel p57-binding protein. Co-immunoprecipitation and reporter gene assays were used to elucidate the physiological and functional relevance of p57/FHL2 interaction. We found in cancer cells that endogenous p57 and FHL2 are in a complex. We observed a substantial induction of established FHL2regulated gene promoters by p57 in reporter gene experiments and detected strong induction of the intrinsic transactivation activity of FHL2. Treatment of cells with histone deacetylase (HDAC) inhibitors and binding of exogenous FHL2 to HDACs indicated repression of FHL2 transcription activity byHDACs. In the presence of the HDAC inhibitor sodium butyrate activation of FHL2 by p57 is abrogated suggesting that p57 shares a common pathway with HDAC inhibitors. p57 competes with HDACs for FHL2 binding which might partly explain the mechanism of FHL2 activation by p57. These results suggest a novel function of p57 in transcription regulation.In order to gain more insight into novel functions of p57, we aimed to identify novel p57 binding partners. Therefore, we performed a yeast two-hybrid screen and obtained the protein FHL2 as a novel p57-interactor.FHL2 is a multifunctional LIM domain only protein which binds cellular proteins via its LIM domains and thereby regulates various cellular processes 18 . Although FHL2 does not directly bind to DNA, it modulates the activity of several transcription factors 19,20 . FHL2 was first described to bind to the hormone-activated androgen receptor (AR) which increases the activity of AR-dependent reporter genes 21 . FHL2 is expressed in the cytoplasm and the nucleus. Interestingly, in several cancer types high levels of nuclear FHL2 correlate with disease progression towards a malignant state. This indicates that FHL2 dependent transcription contributes to cancer development and progression 22,23 .Here we report that p57 strongly activates FHL2 transactivation function and induces the activity of known FHL2-regulated promoters. We provide experimental evidence supporting the hypothesis that FHL2 is repressed by HDACs and p57 relieves this repression by competing with HDACs for FHL2-binding. FHL2 and p57 might regulate transcription as components of chromatin remodeling complexes. Materials and Methodsplasmids and oligonucleotide sequences. Detailed descriptions of novel plasmid constructs, including cloning strategies and sequences of oligonucleotides used are presented in Supplementary inf...
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