it including anaplastic astrocytomas and glioblastomas

To find out whether CDK1 and CDK2 may phosphorylate EZH2 at Thr 350 in vivo, an antibody specific to phosphorylated Thr 350 was raised AZD3463 and pure. The antibody reacted with wild-type however, not EZH2T350A in both 293T and prostate cancer LNCaP cells. This response was blocked by peptide containing the phosphorylated Thr 350, but not by the similar nonphosphorylated peptide. Treatment of cellular proteins with protein phosphatase totally eliminated the reaction of this antibody with EZH2, confirming that the zero Thr 350 S 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, however 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 of endogenous CDK1 and CDK2, and this effect was increased by further treatment with the CDK inhibitor, roscovitine. Lymphatic system Thr 350 phosphorylation of both endogenous and ectopically expressed EZH2 in 293T cells was confirmed by mass spectrometry analysis. Moreover, Thr 350 phosphorylated EZH2 was invariably co localised with all 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 suggest that CDKs can phosphorylate EZH2 at Thr 350 under various physiological and pathological conditions. The biological function of EZH2 is primarily reflected by its international repression of gene transcription7,11. Therefore, we performed microarray analysis to gain molecular insights into the effectation of EZH2 Thr 350 phosphorylation on gene expression in mammalian cells. Endogenous EZH2 was knocked down by an EZH2 particular siRNA, or renewed to physiological levels by ectopically Lonafarnib 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 conducted in LNCaP cells treated using the CDK inhibitor, roscovitine. Additionally, it has been shown previously that histone deacetylase proteins can physically interact with the PRC2 complex23, and treatment of cells with the HDAC inhibitor trichostatin prevents EZH2 mediated gene silencing7,23. Consequently, as positive control, we also performed microarray analysis of LNCaP cells treated with TSA. As shown in Figure 3a, large group of genes were transcriptionally derepressed by EZH2 repressed and knockdown again in tissue with the restored expression of wild-type EZH2.