Study Of CDK11-interacting Protein And The Regulation Role Of Cyclin D3/CDK11 On Androgen Receptor-mediated Transactivation

Cyclin-dependent kinase 11 (p58) (CDK11^p58, p58^PITSLRE, p58^GTA, p58^clk-1) is the earliest identified CDK11 family protein. It was obtained originally through purification of beta-1,4-galactosyltransferase 1 (GalT1). CDK11^p58 interacts with, phosphorylates and enhances the glycosyltransferase activity of GalT1. CDK11^p58 is uniquely expressed in G2/M phase of the cell cycle. Overexpression of CDK11^p58 leads to the decreased cell growth, change in morphology, disorder in mitosis, and arrest in G2/M phase. CDK11^p58 also enhances cell apoptosis in a kinase-dependent manner. Although CDK11^p58 plays an important role in the regulation of cell growth and apoptosis, its upstream regulators and downstream effectors remain unknown.CDK11^p110, another member of CDK11 family, is reported to be associated with the formation of RNAP II complex, and the splicing factors including RNPS1 and cyclin L. Thus, it is suggested that CDK11 signaling may be involved in the eukaryotic transcription and posttranscriptional modification. We screened human fetal liver cDNA library using the full length of CDK11^p58 as the bait. As a result, we found that the C-terminal fragment of HBO1 (histone acetyltransferase binding to ORC1), an MYST family histone acetyltransferase, interacted with CDK11^p58 in yeast. The interaction between CDK11^p58 and the full length of HBO1 was further confirmed in yeast, through GST-pull down assay in vitro and coimmunoprecipitation in vivo. In consistence with the unique expression of CDK11^p58 in G2/M phase, coimmunoprecipitation of endogenous CDK11^p58 and HBO1 was only detected in G2/M phase-synchronized HeLa cells. CDK11^p58 and HBO1 were colocalized in cell nucleus as sporadic particles. HBO1 was first coloned as a new interacting protein of ORC1 and was found to possess intrinsic histone acetyltransferase (HAT) activity. We found that direct addition of recombinant CDK11^p58 protein in the in vitro HAT activity assay enhanced HBO1 HAT activity significantly. Overexpression of CDK11^p58 resulted in the increased HAT activity of endogenous HBO1 in HeLa cells. As a control, the HAT activity of p300, a well-known HAT and coactivator, remained unaffected by CDK1 l^p58 in vitro and in vivo. The above data suggest that CDK11^p58 may regulate the transcription of certain target genes through enhancing HBO1 HAT activity to inhibit cell cycle or promote apoptosis.According to the fact that HBO1 is a corepressor of AR, and that cyclin D3 is also involved in the regulation of transcription mediated by vitamin D receptor (VDR) or activating transcription factor 5 (ATF5), and that cyclin Dl is well identified as a corepressor of AR, we hypothesize that cyclin D3/CDK11^p58 signaling may also play an important role in AR-mediated transcription. We found that CDK11^p58 interacted with AR in vitro and in mammalian cells independent of HBO1. AR is structurally divided into four independent functional domains: transcription activation domain (TAD), DNA-binding domain (DBD), hinge region and ligand-binding domain (LBD). CDK11^p58 contains a Ser/Thr protein kinase domain in the middle of the molecule, an N-terminal domain and a C-terminal domain with unknown function. We constructed the deletion mutants of the two proteins based on their functional domains. Coimmunoprecipitation assay showed that the interacting domains of the two proteins were mapped to the transacription activation unit 1 (TAU1) located in AR TAD, and the kinase domain of CDK11^p58.Androgen/AR signaling is essential for the development and differentiation of male productive and non-productive systems, and is also closely related to the carcinogenesis of male tumors such as prostate cancer. Similar to other nuclear receptors, upon binding to androgen, AR is translocated into nucleus, binds to the promoters, and mediates transcription of target genes. Using the AR reporter gene MMTV-LUC, we found that AR-mediated transactivation was inhibited by CDK11^p58 in a dose-dependent manner. This repressive effect of CDK11^p58 on AR is specific because CMV-LUC which is driven by a CMV promoter is not affected by CDK11^p58. Consistently, PSA-LUC and ARE-LUC, the other two AR reporter genes, are also inhibited by CDK11^p58. The repressive role of CDK11^p58 on AR is HBO1-independent. Interestingly, although CDK11^p58 is encoded by the same mRNA as CDK11^p110, and is structurally located to the C-terminal region of CDK11^p110, the two CDK11 isoforms play distinct roles in AR regulation. CDK11^p110 did not interact with AR in cells, but enhanced the transcriptional activity of AR markedly.In order to clarify whether CDK11^p58 mediated AR regulation is dependent on its kinase activity, we constructed D224N, the kinase-deficient point mutant of CDK11^p58. We found that D224N failed to bind to and repress AR, but enhanced AR-mediated transactivation remarkably. Cyclin D3 promoted the kinase activity of CDK11^p58, and repressed the transcriptional activity of AR in synergy with CDK11^p58, while the kinase activity and repressive effect of CDK11^p58 were both lost when cyclin D3-targeted RNAi was used. Cyclin D3 interacted with AR in vitro and in vivo. Cyclin D3, CDK11^p58 and AR may form a triple complex in vivo. To elucidate the molecular mechanism by which CDK11^p58 repressesAR function, the following possible patterns were considered: 1) AR expression, 2) AR nuclear translocation, 3) AR interaction with coactivators/corepressers,4) binding of AR to ARE, 5) AR N/C interaction, and 6) direct targeting of CDK11^p58 to the AR TAD phosphorylation. We found that neither the expression, nor nuclear translocation, nor the interaction of AR with coregulators, nor the interaction of AR N/C-terminal domains was altered. But the binding of AR to its responsive element was inhibited by CDK11^p58. The in vitro kinase activity assay revealed that AR TAD and TAU1 was phosphorylated by CDK11^p58. Overexpression of CDK11^p58 led to the increased phosphorylation of AR in vivo. In the presence of cyclin D3 RNAi, CDK11^p58-mediated AR phosphorylation was quenched. Androgen/AR signaling is essential for the viability of prostate cancer cells. Cyclin D/CDK11^p58 complex is capable of promote the apoptosis of prostate cancer cells both in LNCaP cells or AR-overexpressing PC-3 cells, while the kinase-deficiently mutant D224N protected cells from apoptosis.In summary, we here report that cyclin D3/CDK11^p58 signaling is involved in the transcriptional regulation in eukaryotic cells as well as the cell cycle regulation.