Androgen Receptor Nuclear Translocation Is Facilitated by the f-Actin Cross-Linking Protein Filamin

Abstract

The human androgen receptor (hAR) is a ligand-dependent transcription factor responsible for the development of the male phenotype. The mechanism whereby nuclear translocation of the hAR is induced by its natural ligand 5alpha-dihydrotestosterone is a phenomenon not fully understood. The two-hybrid interaction trap assay has been used to isolate proteins that interact with the hAR in an attempt to identify molecules involved in hAR transactivation and movement. We have identified the actin-binding protein filamin, a 280-kDa component of the cytoskeleton, as an hAR interacting protein. This interaction is ligand independent but is enhanced in its presence. The functional significance of this interaction was analyzed using a cell line deficient in filamin via transient expression of a green fluorescent protein-hAR chimera. In filamin-deficient cells this revealed that hAR remained cytoplasmic even after prolonged exposure to synthetic ligand. Nuclear shuttling was restored when this cell line regained wild-type expression of filamin. These data suggest a novel role for filamin, implicating it as an important molecule in AR movement from the cytoplasm to the nucleus.

Hypoxia-induced and calpain-dependent cleavage of filamin A regulates the hypoxic response

“…Similar results were also obtained in FLNA [16][17][18][19][20][21][22][23][24] -expressing cells (from 25% to 68%). These observations clearly indicate that FLNA [16][17][18][19][20][21][22][23][24] is able to recapitulate the effect of full-length FLNA on the intracellular distribution of HIF-1α by increasing the nuclear accumulation of the protein in hypoxia. Furthermore, preventing the generation of the C-terminal fragment of FLNA by deletion of the H1 domain completely abrogated FLNAmediated induction of nuclear accumulation of HIF-1α.…”

“…Although the majority of FLNAinteracting partners are membrane-associated or cytoplasmic proteins, several studies have provided evidence that FLNA interacts with nuclear proteins and participates in their signaling pathways. These proteins include transcription factors such as the androgen receptor, Smads, and FOXC1 (9,(17)(18)(19) and proteins involved in DNA repair [e.g., BRCA2 (20)]. In our study we show that FLNA interacts with HIF-1α and regulates the localization and transactivation of this transcription factor.…”

“…Previous studies have shown that the C-terminal fragment of FLNA is required for the nuclear translocation of the androgen receptor (17), and the nuclear localization of the FLNA CT fragment restores the responsiveness of prostate cancer cells to androgens (21). In addition, wingless-related MMTV integration site 5A (Wnt5A)-driven cell migration has been shown to be dependent on FLNA cleavage (22).…”

“…Hypoxia is known to increase cytosolic calcium (26) and has been shown to up-regulate calpain protease activity in several cell types, such as pulmonary arterial endothelial cells (27). The ability of calpain to cleave FLNA is also regulated by phosphorylation (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Several kinases, including PKA, Pak1, and RSK, as well as phosphatases such as calcineurin, have been proposed to regulate FLNA phosphorylation (10).…”

“…6A). We further generated cell lines stably expressing FLAG-tagged FLNA, ΔH1, or FLNA [16][17][18][19][20][21][22][23][24] , and cells transfected with vector only were used as control. As shown in Fig.…”