CDK9 Regulates AR Promoter Selectivity and Cell Growth through Serine 81 Phosphorylation

Abstract

Previously we determined that S81 is the highest stoichiometric phosphorylation on the androgen receptor (AR) in response to hormone. To explore the role of this phosphorylation on growth, we stably expressed wild-type and S81A mutant AR in LHS and LAPC4 cells. The cells with increased wild-type AR expression grow faster compared with parental cells and S81A mutant-expressing cells, indicating that loss of S81 phosphorylation limits cell growth. To explore how S81 regulates cell growth, we tested whether S81 phosphorylation regulates AR transcriptional activity. LHS cells stably expressing wild-type and S81A mutant AR showed differences in the regulation of endogenous AR target genes, suggesting that S81 phosphorylation regulates promoter selectivity. We next sought to identify the S81 kinase using ion trap mass spectrometry to analyze AR-associated proteins in immunoprecipitates from cells. We observed cyclin-dependent kinase (CDK)9 association with the AR. CDK9 phosphorylates the AR on S81 in vitro. Phosphorylation is specific to S81 because CDK9 did not phosphorylate the AR on other serine phosphorylation sites. Overexpression of CDK9 with its cognate cyclin, Cyclin T, increased S81 phosphorylation levels in cells. Small interfering RNA knockdown of CDK9 protein levels decreased hormone-induced S81 phosphorylation. Additionally, treatment of LNCaP cells with the CDK9 inhibitors, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole and Flavopiridol, reduced S81 phosphorylation further, suggesting that CDK9 regulates S81 phosphorylation. Pharmacological inhibition of CDK9 also resulted in decreased AR transcription in LNCaP cells. Collectively these results suggest that CDK9 phosphorylation of AR S81 is an important step in regulating AR transcriptional activity and prostate cancer cell growth.

Regulation of Androgen Receptor and Prostate Cancer Growth by Cyclin-dependent Kinase 5

“…Ser-81 of the AR N terminus is the most intensely phosphorylated site in response to androgen binding (4). The latest report reveals the relevance of Ser-81 phosphorylation and AR promoter selectivity as well as cell growth (5). Our recent work also shows the increase of Ser-81 phosphorylation of AR in the androgen-independent LNCaP sub-line (6).…”

“…Recent evidence indicates that stromal cell-derived factors cause AR Ser-81 phosphorylation and modulate prostate cancer cell growth through the Erk pathway (36). Other kinases are also reported as candidates for phosphorylating AR Ser-81 site, such as Cdk1, Cdk5 (5,8), and Cdk9 (5). With regard to Cdk5, it has been reported to regulate the transcriptional activity of glucocorticoid receptor through phosphorylation (37).…”

Darwinian Selection in Prostate Cancer and Medical Treatment

“…Mutant ARs (Y267F and Y363F) inhibit Ack1-mediated AR transactivation and chromatin binding. In addition, cyclin-dependent kinase 1 or 9 (CDK1 or 9)-mediated phosphorylation of AR at Ser-81 associated with the AR-chromatin interaction [36]. JNK and p38 kinases phosphorylate AR at Ser-650, regulating the nuclear export of AR [37], whereas dephosphorylation by protein phosphatase 1 regulates the AR transcriptional activity and nuclear localization [38] [39].…”

Androgen Receptor Exon 1 Mutation Causes Androgen Insensitivity by Creating Phosphorylation Site and Inhibiting Melanoma Antigen-A11 Activation of NH2- and Carboxyl-terminal Interaction-dependent Transactivation

“…Phosphorylation is a common posttranslational event in the AR NH 2 -terminal region that impacts function to different extents (53)(54)(55)(56). Some of the known AR NH 2 -terminal phosphorylation sites include Ser-81, 308, and 515 phosphorylated by cyclin-dependent kinases (53,54,(57)(58)(59)(60)(61)(62), Ser-282 and 293 phosphorylated by Aurora-A kinase (63), Ser-94 (53, 54), and Ser-578 (61). None of the documented AR NH 2 -terminal phosphorylation sites has been reported to be a site of a naturally occurring mutation that caused AIS.…”