Identification of a Novel Phosphorylation Site in Human Androgen Receptor by Mass Spectrometry

Zhu, Zhenxi; Becklin, Robert R.; Desiderio, Dominic M.; Dalton, James T.

doi:10.1006/bbrc.2001.5030

Cited by 33 publications

(25 citation statements)

References 28 publications

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“…In response to androgens, p-Ser81 is the most abundant (Lee et al 2007); however, it can occur at low levels of androgen, for example, in LNCaP cells Ser81 is phosphorylated in response to as little as 1 nM DHT, while in C4-2 cells concentrations of between 10 and 100 pM is sufficient . Ser308 was found to be phosphorylated in the presence of androgens using MALDI/TOF-MS (Zhu et al 2001), with mutation of this site to alanine inconsistently increasing transcriptional activity (Gioeli et al 2002), yet cyclin D3/CDK11p58 can phosphorylate this site both in vitro and in vivo to result in inhibition of AR activity (Zong et al 2007), suggesting that this residue may be phosphorylated as a mechanism for receptor down-regulation in the nucleus.…”

Section: Serine Phosphorylationmentioning

confidence: 99%

Regulation of the androgen receptor by post-translational modifications

Coffey

Robson

2012

Journal of Endocrinology

119

100

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The androgen receptor (AR) is a key molecule in prostate cancer and Kennedy's disease. Understanding the regulatory mechanisms of this steroid receptor is important in the development of potential therapies for these diseases. One layer of AR regulation is provided by post-translational modifications including phosphorylation, acetylation, sumoylation, ubiquitination and methylation. While these modifications have mostly been studied as individual events, it is becoming clear that these modifications can functionally interact with each other in a signalling pathway. In this review, the effects of all modifications are described with a focus on interplay between them and the functional consequences for the AR.

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Section: Serine Phosphorylationmentioning

confidence: 99%

Regulation of the androgen receptor by post-translational modifications

Coffey

Robson

2012

Journal of Endocrinology

119

100

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“…However, these determinations were made indirectly. Most recently, Ser-308 was directly identified as a phosphorylation site using mass spectrometry (23). We have completed the most comprehensive analysis of AR phosphorylation to date, covering 81% of the protein and 88% of the serine residues by mass spectrometric analysis.…”

Section: Stoichiometric Changes In Ar Phosphorylation-lncapmentioning

confidence: 99%

“…However, these determinations, although a useful first step, are not definitive because in vitro kinase reactions are often not selective, and mutagenesis can alter the phosphorylations on sites distinct from the ones mutagenized. Recently, Ser-308 was directly identified as a phosphorylation site in baculovirusoverexpressed AR using mass spectrometry (23). This is the only site identified in living cells either by mass spectrometry or by in vivo metabolic labeling.…”

mentioning

confidence: 99%

Androgen Receptor Phosphorylation

Gioeli¹,

Ficarro

Kwiek

et al. 2002

Journal of Biological Chemistry

296

149

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Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of crosstalk between growth factor signaling and androgen in prostate development, physiology, and cancer.The steroid hormone receptors are ligand-activated transcription factors. In addition to regulation by steroids, they are also regulated by post-translational modifications generated by signal transduction pathways. Thus, they function not only as transcription factors but also as nodes that integrate multiple extracellular signals. The human progesterone receptor (PR) 1 is phosphorylated on multiple residues; the basal phosphorylation sites, including serines 81, 102, and 162, are rapidly induced in the presence of steroid (1). However, phosphorylation of serines 102, 294, and 345 in response to hormone is temporally delayed, reaching a maximum at 2 h (2). Phosphorylation of PR on Ser-676 in the hinge region has recently been identified (3); the analogous site in the chicken PR is also phosphorylated. When this site is mutated to alanine, subsaturating levels of hormone show severalfold less transcriptional activity compared with wild type (4). At least seven phosphorylation sites have been identified on the glucocorticoid receptor, and the relative level of phosphorylation of these sites appears to be cell cycle-regulated (5-7). Recent evidence indicates that the phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover (8). Growth factors are known to stimulate the ligand-independent activity of the estrogen receptor through the activation of the mitogen-activated protein kinase (MAPK) cascade and the direct phosphorylation of estrogen receptor by MAPK at Ser-118 (9). Hormone binding also regulates the phosphorylation ...

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“…Thus, serine/ threonine kinases seem to be mediators of AR activation. Androgen treatment not only activates AR via conformational changes but also causes significant phosphorylation of AR (21,27,28), suggesting that kinase cascades are also being activated by androgen. The in vivo phosphorylation sites of AR have recently been identified (21,(27)(28)(29); none of which, however, are consensus phosphorylation sites of ERK and AKT, raising the possibility that other novel kinases are involved.…”

Section: Introductionmentioning

confidence: 99%

“…Androgen treatment not only activates AR via conformational changes but also causes significant phosphorylation of AR (21,27,28), suggesting that kinase cascades are also being activated by androgen. The in vivo phosphorylation sites of AR have recently been identified (21,(27)(28)(29); none of which, however, are consensus phosphorylation sites of ERK and AKT, raising the possibility that other novel kinases are involved. Studies to identify protein kinases associated with or induced by AR resulted in the discovery of several novel kinases: ANPK (30), SPAK (31), cyclin-dependent kinase (CDK) 6 (32), CDK7/CAK (33), and PAK6 (34,35).…”

Section: Introductionmentioning

confidence: 99%

Male Germ Cell–Associated Kinase, a Male-Specific Kinase Regulated by Androgen, Is a Coactivator of Androgen Receptor in Prostate Cancer Cells

Hong¹,

Xia²,

Desai³

et al. 2006

Cancer Research

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Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell-associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen-and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence. (Cancer Res 2006; 66(17): 8439-47)

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Identification of a Novel Phosphorylation Site in Human Androgen Receptor by Mass Spectrometry

Cited by 33 publications

References 28 publications

Regulation of the androgen receptor by post-translational modifications

Regulation of the androgen receptor by post-translational modifications

Androgen Receptor Phosphorylation

Male Germ Cell–Associated Kinase, a Male-Specific Kinase Regulated by Androgen, Is a Coactivator of Androgen Receptor in Prostate Cancer Cells

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