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Calmodulin-Androgen Receptor (AR) Interaction: Calcium- Dependent, Calpain-Mediated Breakdown of AR in Lncapprostatecancer Cells

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Calmodulin-Androgen Receptor (AR) Interaction: Calcium- Dependent, Calpain-Mediated Breakdown of AR in Lncapprostatecancer Cells Research Article Calmodulin-Androgen Receptor (AR) Interaction: Calcium- Dependent, Calpain-Mediated Breakdown of AR in LNCaPProstateCancer Cells Ronald P. Pelley,1 Kannagi Chinnakannu,1 Shalini Murthy,1 Faith M. Strickland,2 Mani Menon,1 Q. Ping Dou,3 Evelyn R. Barrack,1 and G. Prem-Veer Reddy1,3 1Vattikuti Urology Institute and 2Department of Dermatology, Henry Ford Hospital; 3Karmanos Cancer Institute and Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan Abstract Introduction Chemotherapy of prostate cancer targets androgen receptor Adenocarcinoma of the prostate is the most frequently (AR) by androgen ablation or antiandrogens, but unfortu- diagnosed cancer and second leading cause of cancer deaths in nately, it is not curative. Our attack on prostate cancer American men (1). Although androgen ablation is the most envisions the proteolytic elimination of AR, which requires a common therapy for disseminated prostate cancer, it is palliative fuller understanding of AR turnover. We showed previously in nature and most patients eventually succumb to hormone- that calmodulin (CaM) binds to AR with important con- refractory disease resistant to chemotherapy. Whether normal or sequences for AR stability and function. To examine the mutated, androgen receptor (AR) is required for growth in both involvement of Ca2+/CaM in the proteolytic breakdown of AR, androgen-sensitive and androgen-insensitive prostate cancer (2). we analyzed LNCaP cell extracts that bind to a CaM affinity Therefore, it is of paramount importance to dissect the various column for the presence of low molecular weight forms of AR ways in which AR is regulated to not simply inactivate but to (intact AR size, f114 kDa). Using an antibody directed against physically eliminate AR (3). This requires understanding the the NH2-terminal domain (ATD) of AR on Western blots, we cellular mechanisms of AR breakdown. identified f76-kDa, f50-kDa, and 34/31-kDa polypeptides in Numerous studies have examined the proteolytic destruction of eluates of CaM affinity columns, suggesting the presence of AR, and our knowledge can at present be divided into three areas. CaM-binding sites within the 31/34-kDa ATD of AR. Under First, AR is degraded to small (V10 residues) peptides by the cell-free conditions in the presence of phenylmethylsulfonyl threonine proteases of the ubiquitin-proteasome system (4–9). fluoride, AR underwent Ca2+-dependent degradation. AR Second, AR can be cleaved by serine proteases (10–16). These degradation was inhibited by N-acetyl-leu-leu-norleu, an studies established that, structurally, AR consists of at least three inhibitor of thiol proteases, suggesting the involvement of domains, the ligand-binding domain (LBD), the DNA-binding calpain. In intact cells, AR breakdown was accelerated by domain (DBD), and the NH2-terminal domain (ATD), separated by raising intracellular Ca2+ using calcimycin, and increased AR protease-sensitive linker regions. Serine proteases cleave AR to breakdown was reversed with the cell-permeable Ca2+ chelator yield a 34-kDa COOH-terminal LBD (12, 17) and the remaining bis-(O-aminophenoxy)-ethane-N,N,N¶,N¶-tetraacetic acid tetra- f76 kDa of AR comprising the ATD and DBD. Other studies (11) (acetoxymethyl)-ester. In CaM affinity chromatography stud- established that proteolysis can cleave AR into two fragments: one ies, the Ca2+-dependent protease calpain was bound to and of f50 kDa containing both the LBD and the DBD and another of eluted from the CaM-agarose column along with AR. Caspase-3, approximately the same size, presumably the ATD (13). Third, the which plays a role in AR turnover under stress conditions, did caspase family of thiol proteases is capable of cleaving AR, not bind to the CaM column and was present in the proenzyme particularly those forms of AR with expanded polyglutamine 2+ form. Similarly, AR immunoprecipitates prepared from whole- repeats (18–22). To date, the cleavage of AR by the Ca -activated, cell extracts of exponentially growing LNCaP cells contained thiol protease calpain has not been described. both calpain and calpastatin. Nuclear levels of calpain and The amount, localization, physiologic functions, and degradation calpastatin (its endogenous inhibitor) changed in a reciprocal of AR are influenced by the following factors. First, AR is transcribed, translated (23, 24), and post-translationally phosphor- fashion as synchronized LNCaP cells progressed from G1 to S phase. These reciprocal changes correlated with changes in ylated (25, 26). Androgen binds to the LBD, inducing conforma- tional change critical to the transcription factor activity of AR as AR level, which increased in late G1 phase and decreased as S phase progressed. Taken together, these observations well as resistance to degradation (17, 27). Following androgen suggest potential involvement of AR-bound CaM in calcium- binding, AR is ready to act as a transcription factor (23), as the DBD controlled, calpain-mediated breakdown of AR in prostate binds to the androgen response element (ARE). The activity of the cancer cells. (Cancer Res 2006; 66(24): 11754-62) DNA-bound complex is governed by binding of coactivator and corepressor proteins to the LBD and the ATD. Finally, AR activity is also controlled by AR-calmodulin (CaM) interaction (28). It is the effect of AR interaction with CaM on the breakdown of AR that we Note: R.P. Pelley, K. Chinnakannu, and S. Murthy contributed equally to this work. examine herein. Requests for reprints: G. Prem-Veer Reddy, Vattikuti Urology Institute, Henry Ford Extending our observation that CaM binds to AR directly (28), Health System, One Ford Place, 2D, Detroit, MI 48202. Phone: 313-874-5991; Fax: 313- we now map the CaM-binding site(s) to the ATD of AR. We show 874-4324; E-mail: [email protected]. 2+ I2006 American Association for Cancer Research. for the first time that Ca stimulates AR breakdown and that CaM doi:10.1158/0008-5472.CAN-06-2918 bound to AR may play an important role in regulating AR levels by Cancer Res 2006; 66: (24). December 15, 2006 11754 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Calpain Cleavage of Androgen Receptor in LNCaP Cells a Sorvall RT7 centrifuge and resuspended in buffer A [0.16 mol/L sucrose, 50 mmol/L Tris-HCl (pH 7.6), 25 mmol/L KCl, 10 mmol/L MgCl2, 70 mmol/L HEPES (pH 7.6), 0.025 mmol/L CaCl2, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2 mmol/L DTT, 1 mmol/L EDTA] at a density of 2 Â 107/mL. The cell suspension was then homogenized in a top-driven Wheaton homogenizer (Wheaton Co., Wheaton, IL) until f90% of the cells were stained with trypan blue dye (usually three strokes at a rotation setting of 3). The cytosolic supernatant was then separated from the nuclear pellet by centrifugation at 4jC in a Beckman Microfuge B at 6,000 rpm for 5 minutes (Beckman Instruments, Palo Alto, CA). A volume of buffer A equal to that used for homogenization was added to the microfuge tube, and the nuclei were then subjected twice to 30 pulses of sonication with a Branson Sonifier 250 (Branson Co., Danbury, CT) set at an output control of 2 and a duty cycle of 20, with intermittent cooling on ice. The sonicated nuclear extract was cleared by centrifugation in the microfuge as described above. Protein concentration in cytosolic and nuclear extracts was assessed with the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) using the Bradford reagent in 96-well plates and read using an EL340 Micro Colorimeter (BioTek Instruments, Winooski, VT). Whole-cell extracts were prepared essentially as described by Cifuentes et al. (28). Briefly, LNCaPcells were suspended at a density of 2 Â 107/mL in buffer A and sonicated and the extracts were clarified as above. Figure 1. AR and its breakdown products in LNCaP cell lysate and UVB irradiation. UV irradiation was administered (31) using a single CaM-agarose affinity column fractions. An f1 mL volume of cell lysate of FS40 lamp (National Biological, Twinsburg, OH). FS40 lamps produce 0.5% 2 Â 107 LNCaP cells (harvested by trypsinization) was applied to 2mL bed UVC (260–280 nm), 62% UVB (280–320 nm), and 38% UVA (320–400 nm) volume columns of CaM-agarose in affinity chromatography buffer containing 2+ drop thru with a peak emission at 313 nm. The average irradiance of the source was 2mmol/L Ca and 1 mmol/L PMSF. After nonadherent material ( ) Â À4 washed through, CaM-binding materials were eluted with buffer lacking Ca2+ approximately 1.58 10 W at 17-cm distance as measured by an IL1700 but containing 10 mmol/L EGTA (EGTA Eluate). Each lane was loaded with Research Radiometer (International Light, Newburyport, MA) with a UVB-1 f20 Ag protein and respectively contained the following: crude cell lysate filter (#25628) and SED 240 detector. LNCaPcells were grown to 70% lane 1 lane 2 ( ), material not binding to CaM, drop thru ( ), and material eluted from confluence in P100 plates. Immediately before UV radiation treatment, the CaM with EGTA (lane 3). After PAGE and transfer, the blots were stained with the pAb N20 and developed with anti-rabbit conjugated antibody as medium was removed from each culture, the cells were washed once with described in Materials and Methods. Data are representative of two experiments, PBS, and fresh PBS was added. Cultures were exposed to one of two doses of each comprising four chromatographic runs. UV radiation (300 J/m2 for 40 seconds or 600 J/m2 for 80 seconds) under sterile conditions with the cover of the culture dish off. Following UV exposure, the PBS was removed and replaced with serum-containing culture engaging calpain/calpastatin in cleavage of AR under physiologic medium and cultured for an additional 12 or 24 hours.
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