Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jun 29;287(27):22959-68.
doi: 10.1074/jbc.M111.338350. Epub 2012 May 14.

Differential regulation of androgen receptor by PIM-1 kinases via phosphorylation-dependent recruitment of distinct ubiquitin E3 ligases

Douglas E Linn  1 , Xi Yang, Yingqiu Xie, Alan Alfano, Dhanraj Deshmukh, Xin Wang, Hermela Shimelis, Hegang Chen, Wei Li, Kexin Xu, Mingyuan Chen, Yun Qiu
Affiliations

Differential regulation of androgen receptor by PIM-1 kinases via phosphorylation-dependent recruitment of distinct ubiquitin E3 ligases

Douglas E Linn et al. J Biol Chem. .

Abstract

Androgen receptor (AR) plays a pivotal role in prostate cancer. Regulation of AR transcriptional activity by post-translational modifications, such as phosphorylation by multiple kinases, is well documented. Here, we report that two PIM-1 kinase isoforms which are up-regulated during prostate cancer progression, namely PIM-1S and PIM-1L, modulate AR stability and transcriptional activity through differentially phosphorylating AR at serine 213 (Ser-213) and threonine 850 (Thr-850). Although both kinases are capable of interacting with and phosphorylating AR at Ser-213, only PIM-1L could phosphorylate Thr-850. We also showed that PIM-1S induced Ser-213 phosphorylation destabilizes AR by recruiting the ubiquitin E3 ligase Mdm2 and promotes AR degradation in a cell cycle-dependent manner, while PIM-1L-induced Thr-850 phosphorylation stabilizes AR by recruiting the ubiquitin E3 ligase RNF6 and promotes AR-mediated transcription under low-androgen conditions. Furthermore, both PIM-1 isoforms could promote prostate cancer cell growth under low-androgen conditions. Our data suggest that these kinases regulate AR stability and transcriptional activity through recruitment of different functional partners in a phosphorylation-dependent manner. As AR turnover has been previously shown to be critical for cell cycle progression in prostate cancer cells, PIM-1 kinase isoforms may promote prostate cancer cell growth, at least in part, through modulating AR activity via distinct mechanisms.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
PIM-1 kinases interact with AR C terminus via their LXXLL motif. A, GST pull-down identifies PIM-1 kinases as candidate AR-interacting proteins. Lysates from CWR-R1 cells were incubated with purified GST-tagged AR C terminus (GST-ARc) overnight and separated by PAGE. Marker (M) shows molecular weights with GST fusion proteins labeled. Bands from stained gel were excised and further analyzed by mass spectrometry. GST tag alone was used as a negative control. B, CWR-R1 cells were infected with lentivirus encoding the shRNA for PIM-1 or the control shRNA. At 48-h postinfection, cell lysates were immunoprecipitated with anti-PIM1 followed by Western blot analysis using the indicated antibodies. C, 293T cells were cotransfected with FLAG-tagged PIM-1 kinases and full-length AR followed by an IP with an anti-AR antibody. Control blots detected input levels of AR and PIM-1 kinase expression. D, 293T cells were cotransfected with AR AF1 (amino acids 1–564) or AF2 (amino acids 622–919) domains and FLAG-tagged PIM-1 constructs. Cell lysates were then immunoprecipitated with an anti-AR antibody followed by immunoblotting with an anti-FLAG antibody. Kinase-inactive mutants are indicated as KM. Total cell lysate levels of AR and FLAG-PIM1 were also immunoblotted as controls. E, 293T cells were transfected to overexpress FLAG-tagged PIM-1 kinase mutant (KM) or LXXLL mutant (LA) constructs. Lysates were incubated with purified GST-tagged AR-AF1 or -AF2 for GST pull-down assay. Western blots of pulldown or input samples were immunoblotted with anti-FLAG. Coomassie Blue-stained (CBS) membrane showed relative loading of GST AR AF constructs.
FIGURE 2.
FIGURE 2.
PIM-1 kinases phosphorylate AR at Ser-213 and Thr-850. A, putative PIM-1 phosphorylation sites in AR protein. B, PIM-1 kinases induce AR phosphorylation at Ser-213. FLAG-tagged PIM-1 kinases were transfected into COS-1 (left) or infected with lentiviruses in LNCaP cells (right). The level of AR Ser-213 phosphorylation in total cell lysates was detected by immunoblotting with phosphospecific antibody for Ser-213 of AR (pARS213). HA-tagged AKT1 served as a positive control for Ser-213 phosphorylation in COS-1 cells. AR, FLAG, tubulin, and HA blots were used as controls. C, COS-1 cells were cotransfected with wild-type AR (AR-WT) or the AR-S213A mutant with FLAG-tagged PIM-1 kinases. Total cell lysates were used for Western blot analysis as in B. D, PIM-1L induces AR phosphorylation at Thr-850. COS-1 cells were transfected with FLAG-tagged PIM-1 kinases and AR (left panel). Lysates were immunoprecipitated with anti-AR followed by immunoblotting with a phospho-threonine (pThr) antibody (right panel). AR and FLAG blots were used as controls. E, a similar IP experiment was performed along with the AR-T850A mutant to confirm specificity of pThr signal PIM-1L-induced AR Thr-850 phosphorylation was detected by an antibody specific for phosphorylated Thr-850 (pT850). F, in vitro kinase assays using purified GST-PIM-1S and -PIM-1L, AR phosphorylation was detected by using pARS213 and pART850 antibodies.
FIGURE 3.
FIGURE 3.
PIM-1S promotes AR turnover by recruitment of Mdm2. A, 293T cells were transfected with AR and FLAG-tagged PIM-1 kinases. At 16 h post-transfection, cells were treated with 10 μg/ml CHX for the times indicated. Total cell lysates were blotted for AR, while FLAG and GAPDH served as controls (top panel). The effects of S213A and T850D mutation on AR stability were also examined as above in cells treated with 100 μg/ml CHX. Quantification of AR levels was carried out using densitometry by normalizing AR to GAPDH expression (bottom panel). Values were set relative to AR expression at time 0. B, LNCaP cells were arrested in G2, M, G1, and S-phases after 16 h treatments of 2 μm etoposide, 200 ng/ml nocodazole, 15 μg/ml lovastatin, or 5 μg/ml aphidicolin, respectively (left panel). Total cell lysates were subjected to Western blot with the antibodies listed. Endogenous AR expression in LNCaP cells was knocked down by lentiviral shRNA and simultaneously replaced with the codon-switched AR-WT or AR-S213A mutant (right panel). Cells were then synchronized in M-phase using nocodazole treatment. AR expression was measured by Western blot with GAPDH serving as a loading control. C, FLAG-tagged PIM-1 kinases were overexpressed in CWR-R1 and LNCaP cells and then maintained in medium containing 0.1% FBS for 24 h. Lysates were subjected to denaturing conditions before IP with anti-AR as described under “Experimental Procedures.” Immunoprecipitates were immunoblotted with anti-Ub antibody while total cell lysate blots were used as controls. D, AR constructs and FLAG-tagged PIM-1 kinases were transfected into COS-1 cells followed by IP with anti-AR. Immunoprecipitates were immunoblotted with Mdm2 while total cell lysates were used for control blots. E, AR constructs and FLAG-tagged PIM-1 kinases were transfected into COS-1 cells followed by IP and Western blot as in C.
FIGURE 4.
FIGURE 4.
PIM-1L enhances AR transcriptional activity via recruitment of RNF6. A, COS-1 cells were cotransfected with FLAG-tagged PIM-1 kinases, HA-tagged RNF6, and AR constructs. Lysates were subjected to denaturing conditions before an IP using an AR antibody. Immunoprecipitates were immunoblotted with HA and Ub antibodies. TCL: total cell lysates. B, effects of PIM-1 kinases on AR regulated ARR2 reporter. AR and FLAG-tagged PIM-1 kinases were transfected into COS-1 cells with the ARR2-LUC reporter. Approximately 24 h post-transfection, cells were maintained in medium containing 0.1% FBS overnight and the effects of PIM-1 kinases on AR transcriptional activity were determined by luciferase assays. Data is shown relative to AR transfected alone whose value was set at 1. *p < 0.01. C, wild type AR and AR mutants were cotransfected into COS-1 cells with FLAG-tagged PIM-1L and luciferase activity of ARR2 reporter measured as in B. Data are shown relative to AR transfected alone whose value was set at 1. *, p < 0.01. D. LNCaP cells were infected with lentiviruses encoding FLAG-tagged PIM-1 kinases or vector control. Approximately 24 h postinfection, cells were maintained in media containing 0.1% FBS and total RNAs were collected 18 h later. The level of AR target genes KLK2, POV1, and PSA were detected by real time RT-PCR (left panel). Data are shown relative to vector control infection whose value was set at 1. *, p < 0.01. The recruitment of AR to the KLK2 and PSA promoter regions was examined using ChIP assays (right panel). The value was normalized with IgG and input level. Data were shown relative to vector control infection whose value was set at 1. *, p < 0.01.
FIGURE 5.
FIGURE 5.
PIM-1 kinases promote prostate cancer growth. A, LNCaP cells were infected with lentiviruses encoding PIM-1 kinases and maintained in medium containing 0.1% FBS (top panel) or 5% charcoal-stripped serum supplemented with 50 pm R1881 (bottom panel) for 6 days. Cell cultures were then fixed and stained using Coomassie Blue dye. B, LNCaP cells were infected with the indicated lenti-virus. At 24 h postinfection cells were cultured in CS medium containing 0.1 nm DHT. Cell proliferation was measured using the CCK8 assays. The experiments were repeated three times and the representative data were shown. C, LNCaP cells were infected with lentiviruses encoding control shRNA or target specific for PIM-1. Cells were grown for 6 days in complete media and stained with Coomassie Blue dye. D, proposed model of the effects of PIM-1 kinases on modulating AR function. PIM-1S induces phosphorylation at serine 213, a residue, which can also be targeted by PIM-1L. Phosphorylation of Ser-213 can promote AR turnover mediated by Mdm2 and cell cycle progression. PIM-1L is able to induce phosphorylation at an addition residue Thr-850 in the ligand binding domain. Phosphorylation of Thr-850 can stabilize AR protein and enhance its binding to RNF6 to promote its transcriptional activity in regulating expression of a subset target genes.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • Urolithin A analog inhibits castration-resistant prostate cancer by targeting the androgen receptor and its variant, androgen receptor-variant 7.
    Chandrasekaran B, Tyagi A, Saran U, Kolluru V, Baby BV, Chirasani VR, Dokholyan NV, Lin JM, Singh A, Sharma AK, Ankem MK, Damodaran C. Chandrasekaran B, et al. Front Pharmacol. 2023 Mar 3;14:1137783. doi: 10.3389/fphar.2023.1137783. eCollection 2023. Front Pharmacol. 2023. PMID: 36937838 Free PMC article.
  • Characterizing prostate cancer risk through multi-ancestry genome-wide discovery of 187 novel risk variants.
    Wang A, Shen J, Rodriguez AA, Saunders EJ, Chen F, Janivara R, Darst BF, Sheng X, Xu Y, Chou AJ, Benlloch S, Dadaev T, Brook MN, Plym A, Sahimi A, Hoffman TJ, Takahashi A, Matsuda K, Momozawa Y, Fujita M, Laisk T, Figuerêdo J, Muir K, Ito S, Liu X; Biobank Japan Project; Uchio Y, Kubo M, Kamatani Y, Lophatananon A, Wan P, Andrews C, Lori A, Choudhury PP, Schleutker J, Tammela TLJ, Sipeky C, Auvinen A, Giles GG, Southey MC, MacInnis RJ, Cybulski C, Wokolorczyk D, Lubinski J, Rentsch CT, Cho K, Mcmahon BH, Neal DE, Donovan JL, Hamdy FC, Martin RM, Nordestgaard BG, Nielsen SF, Weischer M, Bojesen SE, Røder A, Stroomberg HV, Batra J, Chambers S, Horvath L, Clements JA, Tilly W, Risbridger GP, Gronberg H, Aly M, Szulkin R, Eklund M, Nordstrom T, Pashayan N, Dunning AM, Ghoussaini M, Travis RC, Key TJ, Riboli E, Park JY, Sellers TA, Lin HY, Albanes D, Weinstein S, Cook MB, Mucci LA, Giovannucci E, Lindstrom S, Kraft P, Hunter DJ, Penney KL, Turman C, Tangen CM, Goodman PJ, Thompson IM Jr, Hamilton RJ, Fleshner NE, Finelli A, Parent MÉ, Stanford JL, Ostrander EA, Koutros S, Beane Freeman LE, Stampfer M, Wolk A, Håkansson N, Andriole GL, Hoover RN, Machiela MJ, Sørensen KD, Borre M, Blot … See abstract for full author list ➔ Wang A, et al. Nat Genet. 2023 Dec;55(12):2065-2074. doi: 10.1038/s41588-023-01534-4. Epub 2023 Nov 9. Nat Genet. 2023. PMID: 37945903 Free PMC article.
  • International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.
    Burris TP, de Vera IMS, Cote I, Flaveny CA, Wanninayake US, Chatterjee A, Walker JK, Steinauer N, Zhang J, Coons LA, Korach KS, Cain DW, Hollenberg AN, Webb P, Forrest D, Jetten AM, Edwards DP, Grimm SL, Hartig S, Lange CA, Richer JK, Sartorius CA, Tetel M, Billon C, Elgendy B, Hegazy L, Griffett K, Peinetti N, Burnstein KL, Hughes TS, Sitaula S, Stayrook KR, Culver A, Murray MH, Finck BN, Cidlowski JA. Burris TP, et al. Pharmacol Rev. 2023 Nov;75(6):1233-1318. doi: 10.1124/pharmrev.121.000436. Epub 2023 Aug 16. Pharmacol Rev. 2023. PMID: 37586884 Free PMC article. Review.
  • Conditional transgenic expression of PIM1 kinase in prostate induces inflammation-dependent neoplasia.
    Narlik-Grassow M, Blanco-Aparicio C, Cecilia Y, Perez M, Muñoz-Galvan S, Cañamero M, Renner O, Carnero A. Narlik-Grassow M, et al. PLoS One. 2013;8(4):e60277. doi: 10.1371/journal.pone.0060277. Epub 2013 Apr 2. PLoS One. 2013. PMID: 23565217 Free PMC article.
  • Implications of ubiquitin ligases in castration-resistant prostate cancer.
    Qi J, Fan L, Hussain A. Qi J, et al. Curr Opin Oncol. 2015 May;27(3):172-6. doi: 10.1097/CCO.0000000000000178. Curr Opin Oncol. 2015. PMID: 25811345 Free PMC article. Review.
See all "Cited by" articles

References

    1. Chen Y., Sawyers C. L., Scher H. I. (2008) Targeting the androgen receptor pathway in prostate cancer. Curr. Opin. Pharmacol. 8, 440–448 - PMC - PubMed
    1. Debes J. D., Tindall D. J. (2004) Mechanisms of androgen-refractory prostate cancer. N. Engl. J. Med. 351, 1488–1490 - PubMed
    1. McPhaul M. J. (2003) Factors that mediate and modulate androgen action. J. Investig. Dermatol. Symp. Proc. 8, 1–5 - PubMed
    1. Culig Z., Hobisch A., Bartsch G., Klocker H. (2000) Androgen receptor–an update of mechanisms of action in prostate cancer. Urol. Res. 28, 211–219 - PubMed
    1. Marcelli M., Cunningham G. R. (1999) Hormonal signaling in prostatic hyperplasia and neoplasia. J. Clin. Endocrinol. Metab. 84, 3463–3468 - PubMed

Publication types

MeSH terms