Identification of three proline-directed phosphorylation sites in the human androgen receptor.

Zhou, Zhong Xun; Kemppainen, Jon; Wilson, Elizabeth M.

doi:10.1210/mend.9.5.7565807

Cited by 68 publications

(69 citation statements)

References 31 publications

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“…Higher vertebrates gain the ability to regulate the negative charge by phosphorylation. This is also suggested by the human AR phosphorylation site Ser 650 (8,10), which is Asp in lower species. Similarly, the human estrogen receptor-␣ phosphorylation site Ser 118 (67,68) is Asp in the lamprey estrogen receptor (4).…”

Section: Discussionmentioning

confidence: 64%

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An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

He¹,

Bai²,

Hnat³

et al. 2004

Journal of Biological Chemistry

122

113

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The NH 2 -terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH 2 -terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH 2 -terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH 2 -terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH 2 -terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH 2 -terminal domains and other intrinsically unstructured transcriptional regulatory proteins.Steroid receptors depend on multiple domains for their function as ligand-dependent transcriptional activators. The DNAand ligand-binding domains have been studied extensively and have a high degree of structural and functional conservation. In contrast, the largely unstructured NH 2 -terminal domains (1-3) are highly variable in size and sequence, and the molecular mechanisms that contribute to transactivation are not well understood. The size of the NH 2 -terminal region of steroid receptors increases with evolutionary expansion (4, 5), from estrogen receptor-␣ (185 amino acid residues) to the glucocorticoid receptor (GR 1 ; 420 residues), androgen receptor (AR; 558 residues), progesterone receptor (B form; 566 residues), and mineralocorticoid receptor (602 residues). In contrast, transcription factors such as p53, NF-B, and VP16 typically have transcriptional activation domains of Ͻ100 residues.

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Section: Discussionmentioning

confidence: 64%

“…the AR NH 2 -terminal conserved motif; lanes [5][6][7][8][9], and GST-AR-(220 -270m4) (with the L237A/K239M/V241A/V243A mutations; lanes 9 -12). 30% Input (lanes 13-16) represents 30% of total 35 3), and pCMV5-CHIP⌬TPR (lanes 5 and 6).…”

Section: Fig 3 Interaction Of Chip With the Ar Nh 2 -Terminal Consementioning

confidence: 99%

An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

He¹,

Bai²,

Hnat³

et al. 2004

Journal of Biological Chemistry

122

113

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“…Previous studies have shown that AR Ser-81 is phosphorylated in response to androgens (11), but transient transfection studies in AR-negative cell lines indicate that this site is not required for AR transcriptional activity (13). Here we identify cyclindependent kinase 1 (Cdk1, also called Cdc2) as an AR Ser-81 kinase.…”

mentioning

confidence: 78%

“…Ligand binding induced AR phosphorylation at multiple sites (primarily N-terminal Ser͞Pro sites), but the kinases mediating AR phosphorylation and their importance for AR function have not been established (11,13,43,44). Transfected Cdk1 stimulated AR phosphorylation at Ser-81 and increased AR protein expression, whereas Cdk1 inhibitors decreased Ser-81 phosphorylation of the endogenous AR in LNCaP PCa cells and similarly decreased AR protein expression and transcriptional activity.…”

Section: Discussionmentioning

confidence: 99%

“…Cdk1 is a candidate kinase for multiple Ser͞Pro sites in addition to Ser-81 that are phosphorylated in the AR N terminus (11,12,43,44), but mutagenesis results indicated that none of these sites were essential for Cdk1-mediated AR stabilization. One interpretation of these data is that AR can be stabilized by Cdk1-mediated phosphorylation at any one of multiple Ser͞Pro sites in the AR.…”

Section: Discussionmentioning

confidence: 99%

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Androgen receptor phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 1

Chen

Yuan

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

185

203

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Androgen receptors (ARs) are phosphorylated at multiple sites in response to ligand binding, but the kinases mediating AR phosphorylation and the importance of these kinases in AR function have not been established. Here we show that cyclin-dependent kinase 1 (Cdk1) mediates AR phosphorylation at Ser-81 and increases AR protein expression, and that Cdk1 inhibitors decrease AR Ser-81 phosphorylation, protein expression, and transcriptional activity in prostate cancer (PCa) cells. The decline in AR protein expression mediated by the Cdk inhibitor roscovitine was prevented by proteosome inhibitors, indicating that Cdk1 stabilizes AR protein, although roscovitine also decreased AR message levels. Analysis of an S81A AR mutant demonstrated that this site is not required for transcriptional activity or Cdk1-mediated AR stabilization in transfected cells. The AR is active and seems to be stabilized by low levels of androgen in ''androgen-independent'' PCas that relapse subsequent to androgen-deprivation therapy. Significantly, the expression of cyclin B and Cdk1 was increased in these tumors, and treatment with roscovitine abrogated responses to low levels of androgen in the androgen-independent C4-2 PCa cell line. Taken together, these findings identify Cdk1 as a Ser-81 kinase and indicate that Cdk1 stabilizes AR protein by phosphorylation at a site(s) distinct from Ser-81. Moreover, these results indicate that increased Cdk1 activity is a mechanism for increasing AR expression and stability in response to low androgen levels in androgen-independent PCas, and that Cdk1 antagonists may enhance responses to androgen-deprivation therapy.

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Phosphorylation of the androgen receptor at Ser81 is co‐sustained by CDK1 and CDK9 and leads to AR‐mediated transactivation in prostate cancer

et al. 2021

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Androgen receptor (AR) is the principal molecule in prostate cancer (PCa) etiology and therapy. AR re‐activation still remains a major challenge during treatment of castration‐resistant prostate cancer (CRPC) tumors that relapse after castration therapies. Recent reports have indicated the enrichment of Ser81‐phosphorylated AR (pS81) in the nucleus of CRPC cells, and CDK1 and CDK9 as the kinases phosphorylating AR at S81. In the current study we showed that pS81 is preferentially localized in the nucleus in both rapid biopsy metastatic CRPC samples and PCa xenografts, and nuclear pS81 localization is correlated with AR transactivation in tumor xenografts. Chromatin immunoprecipitation (ChIP) analysis demonstrated an alignment of S81 phosphorylation and AR‐mediated transactivation with the chromatin locus openness. Moreover, pS81‐specific ChIP‐Seq showed a disproportional occupancy of pS81 on AR‐activated promoters, while 3C‐ChIP assays further indicated an enrichment of pS81 at the PSA enhancer‐promoter loop, a known AR activating hub. In the latter, CDK9 was shown to modulate the transactivation of the AR and RNA Pol II. Indeed, ChIP and re‐ChIP assays also confirmed that AR‐dependent activation of the PSA enhancer and promoter mediated by pS81 was coupled with activation of Pol II and the pTEFb complex. Mechanistically, we determined that CDK1 and CDK9 sustained the pS81 AR modification in the soluble and chromatin‐bound fractions of PCa cells, respectively. Finally, we demonstrated that CDK1 activity was maintained throughout the cell cycle, and that CDK1 inhibitors restored androgen sensitivity in CRPC tumor cells. Based on these findings, CDK1 and CDK9 could be targeted as pS81 kinases in patients with CRPC, either alone or in conjunction with direct AR antagonists.

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Identification of three proline-directed phosphorylation sites in the human androgen receptor.

Cited by 68 publications

References 31 publications

An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

Androgen receptor phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 1

Phosphorylation of the androgen receptor at Ser81 is co‐sustained by CDK1 and CDK9 and leads to AR‐mediated transactivation in prostate cancer

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