A previous study by our group showed that the ex pression of bone morphogenetic

To determine whether CDK1 and CDK2 could phosphorylate EZH2 at Thr 350 in vivo, an antibody specific to phosphorylated Thr 350 was raised and purified. The antibody reacted with wild type although not EZH2T350A in both 293T and prostate AZD3839 cancer LNCaP cells. This reply was blocked by peptide containing the phosphorylated Thr 350, however, not by the equivalent nonphosphorylated peptide. Treatment of cellular proteins with protein phosphatase totally eliminated the reaction of this antibody with EZH2, verifying that the anti Thr 350 P antibody is specific to phosphorylated Thr 350. Ectopic expression of CDK1 cyclin B1 or CDK2 cyclin E considerably increased Thr 350 phosphorylation of both endogenous and exogenous wild-type EZH2, although not EZH2T350A, in LNCaP cells. Thr 350 phosphorylation of EZH2 was inhibited in cells overexpressing the CDK inhibitors, p21WAF1 and p27KIP1. Thr 350 phosphorylation of endogenous EZH2 was significantly Urogenital pelvic malignancy decreased by knockdown of endogenous CDK1 and CDK2, and this effect was enhanced by further treatment together with the CDK inhibitor, roscovitine. Moreover, Thr 350 phosphorylated EZH2 was often company local together with the proliferation marker Ki 67 in human prostate tumours. We also found that CDK1 and CDK2 connect to EZH2 in vitro and in vivo. These data reveal that CDKs may phosphorylate EZH2 at Thr 350 under different physiological and pathological situations. The biological purpose of EZH2 is mostly reflected by its international repression of gene transcription7,11. Hence, Marimastat we performed microarray analysis to gain insights into the effect of EZH2 Thr 350 phosphorylation on gene-expression in mammalian cells. Endogenous EZH2 was knocked-down by an EZH2 specific siRNA, or restored to physiological levels by ectopically expressing siRNA resistant wild-type EZH2 or siRNA resistant EZH2T350A mutant in LNCaP cells. mRNA samples were then obtained for oligonucleotide microarray profiling research. For contrast, microarray analysis was done in LNCaP cells treated with all the CDK inhibitor, roscovitine. Additionally, it's been shown earlier that histone deacetylase proteins can physically interact with the PRC2 complex23, and treatment of tissue with the HDAC inhibitor trichostatin prevents EZH2 mediated gene silencing7,23. As demonstrated in Figure 3a, huge pair of genes were transcriptionally derepressed by EZH2 knockdown and repressed again in tissues together with the restored expression of wildtype EZH2. Consistent with the role of HDACs in concert with the PRC2 complex7,23, inhibition of HDACs by TSA also led to derepression of the pair of EZH2 target genes.