Cold-inducible RNA-binding protein (CIRP) induces translation of the cell-cycle inhibitor p27Kip1.

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.

Inability to phosphorylate Y88 of p27Kip1 enforces reduced p27 protein levels and accelerates leukemia progression.

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 regulates cell proliferation. Phosphorylation of tyrosine residue 88 (Y88) converts the inhibitor into an assembly factor and activator of CDKs, since Y88-phosphorylation restores activity to cyclin E,A/CDK2 and enables assembly of active cyclin D/CDK4,6. To investigate the physiological significance of p27 tyrosine phosphorylation, we have generated a knock-in mouse model where Y88 was replaced by phenylalanine (p27-Y88F). Young p27-Y88F mice developed a moderately reduced body weight, indicative for robust CDK inhibition by p27-Y88F. When transformed with v-ABL or BCR::ABL1p190, primary p27-Y88F cells are refractory to initial transformation as evidenced by a diminished outgrowth of progenitor B-cell colonies. This indicates that p27-Y88 phosphorylation contributes to v-ABL and BCR::ABL1p190 induced transformation. Surprisingly, p27-Y88F mice succumbed to premature v-ABL induced leukemia/lymphoma compared to p27 wild type animals. This was accompanied by a robust reduction of p27-Y88F levels in v-ABL transformed cells. Reduced p27-Y88F levels seem to be required for efficient cell proliferation and may subsequently support accelerated leukemia progression. The potent downregulation p27-Y88F levels in all leukemia-derived cells could uncover a novel mechanism in human oncogenesis, where reduced p27 levels are frequently observed.

Stimulation of c-Jun/AP-1-Activity by the Cell Cycle Inhibitor p57Kip2.

p57 is a member of the Cip/Kip family of cell cycle inhibitors which restrict the eukaryotic cell cycle by binding to and inhibiting cyclin/CDK complexes. They are considered as tumor suppressors and inactivating genomic mutations of p57 are associated with human overgrowth disorders. Increasing evidence suggests that p57 controls additional cellular processes beyond cell cycle control such as apoptosis, cell migration or transcription. Here we report that p57 can stimulate AP-1 promotor activity. While transactivation by c-Jun is strongly activated by p57, it did not enhance c-Fos induced transcription. This indicates that c-Jun is the target of p57 in the canonical AP-1 heterodimeric transcription factor. We could detect endogenous p57/c-Jun containing complexes in cells by co-immunoprecipitation. The strong stimulation of c-Jun activity is not the consequence of activating phosphorylation in the transactivation domain (TAD) of c-Jun, but rather due to negative interference with c-Jun repressors and positive interference with c-Jun activators. In contrast to full-length p57, the amino- and carboxy-terminal domains of p57 are insufficient for a significant activation of c-Jun induced transcription. When expressed in presence of full length p57, the p57 C-terminus abrogated and the N-terminus enhanced c-Jun activation. This indicates that the C-terminus may bind and sequester a putative activator of c-Jun, whereas the N-terminus may sequester a c-Jun repressor. Interestingly, the p57 aminoterminus is sufficient for binding to the two c-Jun repressors HDAC1 and HDAC3. These data are consistent with a model of c-Jun activation where p57 is a part of large nuclear remodeling/transcription complexes. p57 might stimulate transcription by inhibiting transcription repressor proteins like HDACs via its N-terminus and/or attracting transcription activators through its C-terminus. These data suggest that in addition to its role as a CDK inhibitor and tumor suppressor, p57 may also exert tumor promoting functions by activation of the proto-oncoprotein c-Jun.

The CDK inhibitor p57Kip2 enhances the activity of the transcriptional coactivator FHL2.

The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57Kip2 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 FHL2-regulated 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 by HDACs. 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.

Oncostatin M induces growth arrest by inhibition of Skp2, Cks1, and cyclin A expression and induced p21 expression.

Oncostatin M has been characterized as a potent growth inhibitor for various tumor cells. Oncostatin M-treated glioblastoma cells cease proliferation and instigate astrocytal differentiation. The oncostatin M-induced cell cycle arrest in G(1) phase is characterized by increased level of the cyclin-dependent kinase (CDK) inhibitory proteins p21(Cip1/Waf1/Sdi1) and p27(Kip1). Induction of p21 protein corresponds to increased mRNA level, whereas p27 accumulates due to increased stability of the protein. Interestingly, stabilization of p27(Kip1) occurs even in S phase, showing that p27 stabilization is a direct consequence of oncostatin M signaling and not a result of the cell cycle arrest. Degradation of p27 in late G(1) and S phase is initiated by the ubiquitin ligase complex SCF-Skp2/Cks1. Oncostatin M inhibits expression of two components of this E3 ligase complex (Skp2 and Cks1). Although combined overexpression of Skp2 and Cks1 rescues p27 degradation in S phase, it can not override p27 accumulation in G(1) phase and cell cycle arrest by oncostatin M. In addition to increasing Cdk inhibitor level, oncostatin M also impairs cyclin A expression. Cyclin A mRNA and protein level decline shortly after oncostatin M addition. The accumulation of two CDK inhibitor proteins and the repression of cyclin A expression may explain the broad and potent antiproliferative effect of the cytokine.

Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation.

Statins, such as lovastatin, can induce a cell cycle arrest in the G1 phase. This robust antiproliferative activity remains intact in many cancer cells that are deficient in cell cycle checkpoints and leads to an increased expression of CDK inhibitor proteins p27Kip1 and p21Cip1. The molecular details of this statin-induced growth arrest remains unclear. Here we present evidence that lovastatin can induce the degradation of Skp2, a subunit of the SCFSkp2 ubiquitin ligase that targets p27Kip1 and p21Cip1 for proteasomal destruction. The statin-induced degradation of Skp2 is cell cycle phase independent and does not require its well characterised degradation pathway mediated by APC/CCdh1- or Skp2 autoubiquitination. An N-terminal domain preceding the F-box of Skp2 is both necessary and sufficient for its statin mediated degradation. The degradation of Skp2 results from statin induced depletion of geranylgeranyl isoprenoid intermediates of cholesterol biosynthesis. Inhibition of geranylgeranyl-transferase-I also promotes APC/CCdh1- independent degradation of Skp2, indicating that de-modification of a geranylgeranylated protein triggers this novel pathway of Skp2 degradation.

The p27-Skp2 axis mediates glucocorticoid-induced cell cycle arrest in T-lymphoma cells.

Glucocorticoid therapy is an important treatment modality of hematological malignancies, especially T-cell acute lymphoblastic leukemia (T-ALL). Glucocorticoids are known to induce a cell cycle arrest and apoptosis in T-lymphoma cells. We could demonstrate that the cell cycle arrest induced by the synthetic glucocorticoid dexamethasone (Dex) clearly precedes apoptosis in human CEM T-ALL and murine S49.1 T-lymphoma cells. Cyclin D3 is strongly downregulated, whereas the CDK inhibitor p27 (Kip1) (p27) is strongly upregulated in response to dexamethasone in these cells. RNAi-mediated knockdown of p27 as well as overexpression of its negative regulator Skp2 revealed the critical function of p27 in the Dex-induced G 1 arrest of CEM cells. Our studies indicate that several mechanisms contribute to the increase of p27 protein in our T-lymphoma cell lines. We found a significant upregulation of p27 mRNA in S49.1 and CEM cells. In addition, Dex treatment activated the mouse p27 promotor in reporter gene experiments, indicating a transcriptional regulation. However, the relatively moderate induction of p27 mRNA levels by Dex did not explain the strong increase of p27 protein in CEM and S49.1 cells. We found clear evidence for a posttranslational mechanism responsible for the robust increase in p27 protein. Dex treatment of S49.1 and CEM cells increases the half-life of p27 protein, which indicates that decreased protein degradation is the primary mechanism of p27 induction by glucocorticoids. Interestingly, we found that Dex treatment decreased the protein and mRNA levels of the negative regulator of p27 protein and E3 ubiquitin ligase subunit Skp2. We conclude that the cell cycle inhibitor p27 and its negative regulator Skp2 are key players in the glucocorticoid-induced growth suppression of T-lymphoma cells and should be considered as potential drug targets to improve therapies of T-cell malignancies.

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases.

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.

Cell cycle-dependent translation of p27 involves a responsive element in its 5'-UTR that overlaps with a uORF.

p27(Kip1) regulates cell proliferation by binding to and modulating the activity of cyclin-dependent kinases. The CDK inhibitor is haploinsufficient for tumor suppression and reduced p27 activity is fundamental for the development of many human malignancies. Consistently, reduced p27 protein provides independent prognostic information in various tumors including breast, prostate, colon and gastric carcinomas. In normal cells, p27 protein increases in growth arrest but also oscillates during cell cycle progression. Expression of p27 is regulated through mechanisms including transcription, translation and ubiquitin-mediated degradation. Each of these pathways may contribute to deregulation of p27 in hyperproliferative diseases. p27 translation increases in proliferating cells during G(1) phase and declines as cells enter S phase. To investigate the mechanisms of p27 translational control, we analyzed fragments of the p27 transcript for their contribution to cell cycle regulated translation. We found that an element in the p27 5'-UTR can render reporter translation cell cycle sensitive with maximal translation in G1-arrested cells. This novel element of 114 nt contains a G/C-rich hairpin domain that is predicted to form multiple stable stemloops and also overlaps with a small upstream ORF (uORF). Both structures contribute to cell cycle-regulated translation. The uORF can be translated in vitro and its sequence and position are highly conserved in mice and chickens. Interestingly, the precise sequence or the length of the uORF-encoded peptide are not important for p27 translation, consistent with the idea that ribosomal recruitment to its initiation codon rather than the translation product itself contributes to the regulation.

ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5'UTR.

p27Kip1 restrains cell proliferation by binding to and inhibiting cyclin-dependent kinases. To investigate the mechanisms of p27 translational regulation, we isolated a complete p27 cDNA and identified an internal ribosomal entry site (IRES) located in its 5'UTR. The IRES allows for efficient p27 translation under conditions where cap-dependent translation is reduced. Searching for possible regulators of IRES activity we have identified the neuronal ELAV protein HuD as a specific binding factor of the p27 5'UTR. Increased expression of HuD or the ubiquitously expressed HuR protein specifically inhibits p27 translation and p27 IRES activity. Consistent with an inhibitory role of Hu proteins in p27 translation, siRNA mediated knockdown of HuR induced endogenous p27 protein levels as well as IRES-mediated reporter translation and leads to cell cycle arrest in G1.