Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions

Abstract

The androgen receptor (AR), a nuclear transcription factor, mediates male sexual differentiation, and its excessive action is associated with prostate cancer. We have characterized a negative regulatory domain in the AR hinge region, which interacted with filamin A (FLNa), an actin-binding cytoskeletal protein. FLNa interfered with AR interdomain interactions and competed with the coactivator transcriptional intermediary factor 2 to specifically down-regulate AR function. Although full-length FLNa was predominantly cytoplasmic, a C-terminal 100-kDa fragment of FLNa colocalized with AR to the nucleus. This naturally occurring FLNa fragment repressed AR transactivation and disrupted AR interdomain interactions and transcriptional intermediary factor 2-activated AR function in a manner reminiscent of full-length FLNa, raising the possibility that the inhibitory effects of cytoplasmic FLNa may be transduced through this fragment, which can localize to the nucleus and form part of the pre-initiation complex. This unanticipated role of FLNa adds to the growing evidence for the involvement of cytoskeletal proteins in transcription regulation.

Figure 1

Transactivity of AR hinge deletion mutants and effect of FLNa on AR function. (A) The AR consists of a TAD and a DBD, linked to the LBD by a hinge region. The AR hinge (amino acids 622–670) used as bait in the yeast two-hybrid screen is in bold. WT AR or the deletion mutants [(Δ628–646), (Δ647–670), and (Δ622–670)] were tested for their ability to transactivate the ARE₂-Luc reporter in the presence of 1 nM DHT (WT AR set at 100% ± SEM). (B) Structure of monomeric FLNa. Indicated in this schematic representation are the actin-binding domain (ABD), hinge-1 and -2 (H1, H2), and the 24 Ig-like repeats. (C and D) WT AR was coexpressed with increasing amounts of FLNa, and androgenic activity with (filled bars) or without (open bars) 1 nM DHT was measured with ARE₂-Luc (C) or prostate-specific antigen–luciferase (D). The arrow (C Upper) indicates immunoblot analyses of AR protein from representative cell lysates.

Figure 2

Effect of FLNa on AR N–C interaction, and TIF2-mediated coactivation. (A) The two-hybrid system was used to measure the interaction between AR N and C termini. The ARTAD (amino acids 1–565) fused in-frame to the activation domain of VP16 was coexpressed with GAL-ARLBD (628–919) or GAL-ARLBD (647–919), and N–C interactions were measured by using the GAL4-Luc reporter gene with and without 10 nM DHT. (B and C) ARTAD and ARLBD (628–919) (B) or ARLBD (647–919) (C) chimeric proteins were coexpressed with indicated doses (nanograms) of FLNa and 10 nM DHT. N–C interactions were measured with the GAL4-Luc reporter gene. (D–F) Effect of TIF2 on FLNa repression. Increasing amounts of TIF2 were coexpressed with WT AR and FLNa (D). Increasing amounts of FLNa were coexpressed with TIF2 and WT AR (E) or AR(Δ628–646) (F).

Figure 3

Expression of endogenous FLNa fragments and localization of C-terminal fragment of FLNa. (A) Immunoblot of FLNa fragments from HeLa, FLNa-repleted A7, FLNa-deficient M2, prostate carcinoma PC3, and DU145, primary genital skin fibroblasts containing normal AR [LDC, cultured with (+) or without DHT] or nonfunctional mutant AR (TBL) cell cultures. Cell extracts were probed with an antibody that recognizes FLNa repeats 16–20. Arrows indicate full-length FLNa (280 kDa) and a fragment (100 kDa) corresponding to FLNa(16–24). Immunoreactive bands (≈185 and 160 kDa, marked by *) were observed only in extracts from genital fibroblasts. The cytoskeletal paxillin was used to indicate the presence of proteins in each lane. (B) Subcellular localization of GFP-FLNa constructs. Chimeric full-length filamin (FL) or FLNa(16–24, aa1788–2647) linked to GFP was coexpressed with WT AR in the absence (+vehicle) or presence of DHT. Representative green fluorescence images for GFP-FLNa constructs (GFP), red immunofluorescence images for AR (AR), and merged images after superimposition of GFP and AR (merge) are shown. Note that yellow in the merged images indicate colocalization of AR and FLNa(16–24) to the nucleus.

Figure 4

Effect of FLNa(16–24) on AR N–C interaction and TIF2-mediated coactivation. (A) Indicated doses of full-length FLNa or FLNa(16–24) were coexpressed with WT AR in the presence or absence of DHT and androgenic activity measured with ARE₂-Luc. (B) Effect of FLNa(16–24) on AR N–C interaction. Increasing amounts of FLNa(16–24) were coexpressed with chimeric ARTAD and ARLBD and protein–protein interactions measured with GAL4-Luc as in Fig. 2C. (C and D) Effect of TIF2 on FLNa(16–24) repression. Increasing amounts of TIF2 were coexpressed with WT AR and FLNa(16–24) (C). Increasing amounts of FLNa(16–24) were coexpressed with TIF2 and WT AR (D). Transactivation activity was measured with ARE₂-Luc as in Fig. 2 D and E.

Figure 5

Model of FLNa/TIF2-mediated AR transcription. Dimeric FLNa (blue), in the cytoplasm, binds the f-actin cytoskeleton (pink) orthogonally through its N-terminal actin-binding domain (ABD, yellow). AR (orange bars) interacts with the C-terminal end of FLNa through the AR hinge (black loop) and the ARLBD. Cleavage of FLNa at H1 (red loop) releases FLNa(16–24), which colocalizes with the AR into the nucleus. Here, FLNa(16–24) and TIF2 (green) compete for binding to the ARLBD and AR hinge. When the N-terminal subdomain of the AR hinge region is deleted (ΔAR), one of the binding sites for FLNa(16–24) is lost, leading to enhanced TIF2 binding and improved TAD–LBD interactions and consequently increased AR transactivity. CM, NM, cytoplasmic and nuclear membranes.