CDK9 regulates AR promoter selectivity and cell growth through serine 81 phosphorylation.
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.
Gordon
Vicki
V
University of Virginia, Department of Microbiology, Charlottesville, Virginia 22908, USA.
Bhadel
Shriti
S
Wunderlich
Winfried
W
Zhang
JoAnn
J
Ficarro
Scott B
SB
Mollah
Sahana A
SA
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Xenarios
Ioannis
I
Hahn
William C
WC
Conaway
Mark
M
Carey
Michael F
MF
Gioeli
Daniel
D
D059002:Androgen Receptor Antagonists / Q000378:metabolism; D000728:Androgens / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D048708:Cell Growth Processes / Q000235:genetics; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D056906:Cyclin T / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D059002:Androgen Receptor Antagonists; D000728:Androgens; D056906:Cyclin T; D005419:Flavonoids; D010880:Piperidines; D034741:RNA, Small Interfering; D011944:Receptors, Androgen; D012694:Serine; C077990:alvocidib; D004004:Dichlororibofuranosylbenzimidazole; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D012697:Serine Endopeptidases; C421305:TMPRSS2 protein, human