represent only a subset of the multiple molecular mediators of glioma tumorigen

To determine whether CDK1 and CDK2 can phosphorylate EZH2 at Thr 350 in vivo, an antibody specific to phosphorylated Thr 350 grew up and purified. The antibody reacted with wild-type however not EZH2T350A in both 293T and prostate cancer LNCaP cells. This reaction was blocked by peptide containing BAM7 the phosphorylated Thr 350, but not by the matching nonphosphorylated peptide. Treatment of cellular proteins with protein phosphatase totally removed the reaction of this antibody with EZH2, validating that the anti Thr 350 G antibody is specific to phosphorylated Thr 350. Ectopic expression of CDK1 cyclin B1 or CDK2 cyclin E substantially 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 substantially reduced by knockdown Metastasis of endogenous CDK1 and CDK2, and this effect was increased by additional treatment with the CDK inhibitor, roscovitine. Thr 350 phosphorylation of both endogenous and ectopically expressed EZH2 in 293T cells was confirmed by mass spectrometry analysis. Furthermore, Thr 350 phosphorylated EZH2 was often co local together with the proliferation marker Ki 67 in human prostate tumours. We also found that CDK1 and CDK2 interact with EZH2 in vitro and in vivo. These data reveal that CDKs can phosphorylate EZH2 at Thr 350 under various physiological and pathological circumstances. The natural function of EZH2 is generally reflected by its global repression of gene transcription7,11. Thus, we performed NSC 405020 microarray analysis to gain molecular insights in to the aftereffect of EZH2 Thr 350 phosphorylation on gene expression in mammalian cells. Endogenous EZH2 was knocked down by an EZH2 particular siRNA, or repaired 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 analysis. For contrast, microarray analysis was done in LNCaP cells treated with all the CDK inhibitor, roscovitine. Additionally, it has been shown previously that histone deacetylase proteins can actually interact with the PRC2 complex23, and treatment of tissue with the HDAC inhibitor trichostatin blocks EZH2 mediated gene silencing7,23. Therefore, as positive control, we also performed microarray analysis of LNCaP cells treated with TSA. As demonstrated in Figure 3a, huge set of genes were transcriptionally derepressed by EZH2 knockdown and repressed again in cells together with the restored expression of wild-type EZH2.