Inhibitors of Protein Methyltransferases as Chemical Tools

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Rabbit Polyclonal to TRAPPC6A

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. of AR transcriptional activity and AR N-C interaction. Diminished Vav3-Cdc37 interaction also caused decreased prostate cancer cell proliferation selectively in Vav3-expressing cells. Taken together, we identified a novel Vav3 interacting protein that enhances Vav3 co-activation of AR and prostate cancer cell proliferation. Vav3-Cdc37 interaction may provide a new therapeutic target in prostate cancer. benign tissue (5). Higher levels of Vav3 were recently demonstrated in metastatic human prostate cancer specimens, and Vav3 expression in primary disease was shown to predict earlier biochemical recurrence (9). Targeting a constitutively active Vav3 allele to prostate epithelium of transgenic mice results in prostate adenocarcinoma development (10). Consistent with a key 27495-40-5 manufacture role in CRPC, Vav3 enhances AR transcriptional activity and confers robust castration-resistant growth in a tumor xenograft model (4, 11). Vav3 may also participate in other human cancers (12C15). Vav3 overexpression is correlated with poor differentiation of breast cancer and is a predictor of decreased survival in patients with glioblastoma (12). Vav3 also plays a role in the development of anaplastic large cell lymphomas (13). Vav3 is up-regulated in human gastric cancer, and Vav3 overexpression is inversely correlated with gastric cancer patient survival (14). Vav3 and related family members, Vav1 and Vav2, form a subgroup of diffuse B-cell lymphoma (Dbl) GEF proteins. Vav3 activates Rho GTPases by catalyzing the exchange of GDP for GTP 27495-40-5 manufacture (16). Like other Dbl proteins, Vav3 contains a tandem arrangement of the Dbl homology (DH) domain and a pleckstrin homology (PH) domain. The DH domain interacts with Rho proteins and is responsible for catalytic activity. We previously found that GEF deficient Vav3 mutants retain the capacity to enhance androgen-inducible AR activity and AR N-C interaction, a requirement for optimal receptor transcriptional activity (17). However, mutation (W493L) or deletion of the Vav3 PH domain results in failure of Vav3 to co-activate AR. Further, the Vav3 W493L PH domain mutant is largely 27495-40-5 manufacture excluded from the nucleus. Nuclear localization of Vav3 is needed for AR co-activation, and Vav3 is present with AR on androgen response element-containing regions of chromatin (11). To understand in greater detail Vav3 enhancement of AR transcriptional activity in prostate cancer, we searched for novel Vav3 interacting proteins. Because we found that the central region of Vav3 encompassing the DH-PH and cysteine-rich domains (CRD) was sufficient for co-activation of the AR, we used this portion of Vav3 in a Rabbit Polyclonal to TRAPPC6A yeast two-hybrid screen to identify Vav3 binding partners that might participate in AR co-activation. Interestingly, we identified the Hsp90 co-chaperone Cdc37 as a new Vav3 interacting protein. Cdc37 confers Hsp90 specificity for client protein kinases (18C20). In addition to serving as an Hsp90 co-chaperone, Cdc37 appears to also function as a chaperone independent of Hsp90 with client proteins ranging from protein kinases to steroid hormone receptors (21C27). Analysis of publicly available databases and published data reveals that Cdc37 is up-regulated in localized human prostate cancer compared with benign prostate tissues (28). We demonstrate here that Cdc37 interacts with Vav3 in human prostate cancer cells and selectively enhances Vav3 co-activation of AR, AR N-C interaction, and proliferation of Vav3-expressing prostate cancer cells. EXPERIMENTAL PROCEDURES Culture and Chemical Reagents Cell culture media (RPMI 1640 and DMEM) were obtained from Life Science Technologies (Gaithersburg, MD). FBS was obtained from Hyclone Laboratories, Inc. (Logan, UT). The human prostate cancer cell lines LNCaP (ATCC, Manassas, VA, catalog no. CRL 1740; batch F-11701) and PC-3 (ATCC catalog no. CRL 1435; batch F-11154) were cultured in RPMI 1640 supplemented with 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mm l-glutamine 27495-40-5 manufacture (Life Science Technologies), and 10% FBS. The HEK293T (ATCC catalog no. CRL 11268) and COS1 (ATCC catalog no. CRL 1650) were cultured in DMEM supplemented with 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mm l-glutamine (Life Science Technologies), and 10% FBS. The synthetic analog of androgen, R1881, was purchased from PerkinElmer Life Sciences. 5-Bromo-4-chloro-3-indoxyl–d-galactopyranoside was purchased from Gold BioTechnology, Inc. (St. Louis, MO). Plasmids The PSA luciferase (PSA-Luc) reporter plasmid (kindly provided by Dr. Carlos Perez-Stable, University of Miami) consists of the PSA promoter and 5-flanking region, which contain both the distal (?5325 to ?4023) and the proximal (?542 to +12) ARE-containing enhancer regions but lack the intervening sequences. Plasmids pJG4-5 (and reporters.