Phosphorylation of Progesterone Receptor Serine 400 Mediates Ligand-Independent Transcriptional Activity in Response to Activation of Cyclin-Dependent Protein Kinase 2
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Human progesterone receptors (PR) are phosphorylated by cyclin-dependent protein kinase 2 (CDK2) at multiple sites, including Ser400. Herein, we have addressed the significance of phosphorylation of this residue. PR phospho-Ser400-specific antibodies revealed regulated phosphorylation of Ser400 in response to progestins and mitogens, and this correlated with increased CDK2 levels and activity. Expression of cyclin E elevated CDK2 activity and downregulated PR independently of ligand. Similarly, overexpression of activated mutant CDK2 increased PR transcriptional activity in the absence and presence of progestin. Mutation of PR Ser400 to alanine (S400A) blocked CDK2-induced PR activity in the absence, but not in the presence, of progestin. PR was unresponsive to activated CDK2 in breast cancer cells with elevated p27, and RNA interference knock-down of p27 partially restored CDK2-induced ligand-independent PR activation. Similarly, in p27(-/-) mouse embryonic fibroblasts, elevated CDK2 activity increased wild-type (wt) but not S400A PR transcriptional activity in the absence of progestin. CDK2 induced nuclear localization of unliganded wt but not S400A PR; liganded S400A PR exhibited delayed nuclear accumulation. These studies demonstrate that CDK2 regulates PR in the absence of progestins via phosphorylation of Ser400, thus revealing a novel mechanism for upregulated PR transcriptional activity in human breast cancer cells expressing altered cell cycle regulatory molecules.Keywords:
Cyclin E
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Hyperphosphorylation
Retinoblastoma protein
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when CDK2 activity was very low. At 24 h, when CDK2 activity was maximal, the amount of bound- ~27~'p' decreased strongly. The amount of p21P bound to these complexes was low during the first 13 h but subsequently increased. No cyclin A-CDK2 complexes were found during the first 13 h after PH. At 24 h, complexes containing low levels of both in- hibitors were detected and at 28 h, a significant in- crease in p21ciP' and ~27~'p' associated with cyclin A-CDK2 was observed. Conclusions: ~27~'p' acts as a brake on cyclin E- CDK2 activity during the first 13 h after a PH. Both p21Ql and ~27~'r' down-regulate cyclin A-CDK2 activity at 28 h after PH, after its maximal activation.
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Cyclin-dependent kinases (CDKs) play a key role in activating essential cell biology processes including cellular proliferation. Inappropriate regulation of CDKs has been implicated in driving several different forms of cancer. One of the regulatory factors is the need to bind to Cyclin-partners before they can be activated and advance the cell cycle. Cyclins are overexpressed in several different cancers, hence activating their relevant CDK. Hyperactive CDK2 in particular is implicated in many cancers, and while many drugs have shown preclinical promise, none have successfully passed through clinical development. Among the complications of targeting CDK2 is the fact that non-classical cyclin partners from the Speedy/RINGO family of proteins can alter the conformation of the kinase. Using computational approaches, we provide data supporting that the active site of CDK2 differs when bound to Spy1 as compared to classical cyclins. Furthermore, combining computational models with experimental techniques we provide data that many small molecule inhibitors have reduced activity against Spy1-bound CDKs. This work supports the need to develop new inhibitors capable of inhibiting the Spy1-CDK2 complex, and suggests that computational tools can be beneficial toward accomplishing this goal.
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Liver Regeneration
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We examined transforming growth factor-beta 1 (TGF-beta 1) effects on cell cycle progression of human colon carcinoma FET cells. TGF-beta 1 inhibited DNA synthesis and cyclin-dependent kinase (CDK) activity after release from growth arrest in association with induction of the p21 CDK inhibitor, whereas cyclins, CDKs, and p27 protein levels remained relatively unchanged. The decrease in CDK2 kinase activity was the result of increased p21 association with cyclin A-CDK2 and cyclin E-CDK2. TGF-beta 1 treatment in late G(1) showed reduced induction of p21 protein levels in association with increased DNA synthesis. Consequently, p21 induction in early G(1) is critical for TGF-beta 1 inhibition of CDK2 kinase activity. Although TGF-beta 1 treatments in late G(1) failed to induce p21 protein, p21 mRNA induction was observed in late G(1) and in S phase. Further analysis showed that TGF-beta 1 treatment in early G(1) increases p21 protein stability throughout the G(1) and S phases of the cell cycle. Our results demonstrate that TGF-beta 1 stimulation of p21 is regulated at the posttranscriptional and transcriptional levels. This is a novel mechanism of TGF-beta 1 inhibition requiring early G(1) induction and stabilization of p21 protein, which binds to and inhibits cyclin E-CDK2 and cyclin A-CDK2 kinase activity rather than direct modulation of cyclin or CDK protein levels as seen in other systems.
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4409 Cyclin-dependent kinases (CDK) regulate cell cycle progression, and CDK deregulation due to cyclin over-expression, mutation or tumor suppressor gene malfunction is a common feature of human cancer. CDKs are being actively investigated as targets for cancer therapy, and CDK inhibitors have entered clinical trials, e.g. flavopiridol, R-roscovitine and BMS387032. CDK2, in association with cyclins E and A, is involved in G1/S and S phase progression, and molecular pathology studies have implicated CDK2 as a target for drug therapy. However, recent molecular genetic studies in tumor cell lines and in knock-out mouse models have questioned the validity of CDK2 as a target (Tetsu and McCormick Cancer Cell 2003 3:233; Ortega et al., Nature Genetics 2003 35:25). Specifically, mice with homozygous deletion of CDK2 grow normally and give rise to embryonic fibroblasts (MEFs) with equivalent replicative capacity to their WT counterparts. We have recently described the design and synthesis of NU6102 (6-cyclohexylmethoxy-2-(4’-sulfamoylanilino) purine), a potent and selective CDK2 inhibitor (Davies at al., Nature Structural Biology 2002, 9:745). The current study describes the growth inhibitory activity of NU6102 in CDK2 WT and KO MEFs in comparison to the standard compound R-roscovitine. The CDK inhibitory activities of NU6102 and R-roscovitine were defined using a panel of purified human CDKs, ATP concentrations of 12.5 microM (CDK1,2 and 4) or 100 microM (CDK5 and 7), and appropriate substrates. Concentrations (microM) required to inhibit CDKs (CDK1/B; CDK2/A3; CDK4/D; CDK5/p25; CDK7/H) by 50% were: 0.25+/−0.05; 0.005+/−0.001; 1.5+/−0.7; 0.48+/−0.07; 4.4+/−0.8 to NU6102; However, R-roscovitine against these CDKs were 6; 0.41+/−0.05; 5.5+/−1.1; 0.87+/−0.14; 0.84+/−0.04, respectively. Thus NU6102 shows 50-fold selectivity for CDK2, whereas R-roscovitine is less selective. In a 5-day sulphorhodamine B growth inhibition assay, NU6102 had a GI50 value of 9+/−2 microM for CDK2 WT MEFs, whereas there was no growth inhibition up to and including 25 microM in the CDK KO MEFs. At concentrations where NU6102 showed differential activity against the CDK2 WT MEFs (
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