Báo cáo khoa học: Nuclear actin and actin-binding proteins in the regulation of transcription and gene expression docx

Nuclear actin and actin-binding proteins in the regulationof transcription and gene expression Bin Zheng1, Mei Han1, Michel Bernier2 and Jin-kun Wen1 1 Department of Biochemistry and Mol

Nuclear actin and actin-binding proteins in the regulation

of transcription and gene expression

Bin Zheng1, Mei Han1, Michel Bernier2 and Jin-kun Wen1

1 Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, China

2 Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

Actin is a major component of the cytoskeleton and

plays a critical role in all eukaryotic cells The actin

cytoskeleton functions in diverse cellular processes,

including cell motility, contractility, mitosis and

cytoki-nesis, intracellular transport, endocytosis and secretion

[1,2] In addition to these mechanical functions, actin

has also been implicated in the regulation of gene

tran-scription, through either cytoplasmic changes in

cyto-skeletal actin dynamics [3] or the assembly of

transcriptional regulatory complexes [4] Cytoskeletal

actin dynamics, i.e actin polymerization by which monomeric actin (globular actin or G-actin) is assem-bled into long actin polymers (filamentous actin or F-actin) and actin deploymerization by which F-actin

is severed into G-actin, is key for these diverse func-tions The dynamic nature of the actin cytoskeleton

is determined spatiotemporally by the actions of numerous actin-binding proteins (ABPs) The activity

of different classes of ABP controls actin nucle-ation, bundling, filament capping, fragmentation and

Keywords

actin dynamics; actin-binding protein;

chromatin remodeling; gene regulation;

muscle-specific gene; nuclear actin; nuclear

receptor; ribonucleoprotein; RNA

polymerases; transcription complex

Correspondence

J.-k Wen, Department of Biochemistry and

Molecular Biology, Hebei Medical

University, No 361, Zhongshan East Road,

Shijiazhuang 050017, China

Fax: +86 311 866 96180

Tel: +86 311 862 65563

E-mail: wjk@hebmu.edu.cn

(Received 12 January 2009, revised 20

February 2009, accepted 26 February 2009)

doi:10.1111/j.1742-4658.2009.06986.x

Nuclear actin is involoved in the transcription of all three RNA polymerases,

in chromatin remodeling and in the formation of heterogeneous nuclear ribonucleoprotein complexes, as well as in recruitment of the histone modi-fier to the active gene In addition, actin-binding proteins (ABPs) control actin nucleation, bundling, filament capping, fragmentation and monomer availability in the cytoplasm In recent years, more and more attention has focused on the role of actin and ABPs in the modulation of the subcellular localization of transcriptional regulators This review focuses on recent developments in the study of transcription and transcriptional regulation by nuclear actin, and the regulation of muscle-specific gene expression, nuclear receptor and transcription complexes by ABPs Among the ABPs, striated muscle activator of Rho signaling and actin-binding LIM protein regulate actin dynamics and serum response factor-dependent muscle-specific gene expression Functionally and structurally unrelated cytoplasmic ABPs interact cooperatively with nuclear receptor and regulate its transactiva-tion Furthermore, ABPs also participate in the formation of transcription complexes

Abbreviations

ABLIM, actin-binding LIM protein; ABP, actin-binding protein; ANF, atrial natriuretic factor; AR, androgen receptor; CARM1, coactivator-associated arginine methyltransferase 1; CBP, CREB binding protein; DBD, DNA-binding domain; FHL, four and a half LIM domains; FLAP1, Fli-I LRR-associated protein 1; Fli-I, flightless-1; FLNa, filamin-A; FOXC1, forkhead box C1; GRIP1, glucocorticoid receptor-interacting protein 1; HAT, histone acetyltransferase; HDAC, histone deacetylase; HF, hydroxyflutamide; hhLIM, human heart LIM protein; hnRNPs, heterogeneous nuclear ribonucleoproteins; LBD, ligand-binding domain; LEF1⁄ TCF, lymphoid enhancer factor ⁄ T-cell factor; LRR, leucine rich repeat; MEF2, myocyte enhancer factor 2; MRTF, myocardin-related transcription factor; NLS, nuclear localization signals; NM1, nuclear myosin 1; PBX1, pre-B-cell leukemia transcription factor 1; PCAF, p300⁄ CREB binding protein-associated factor; PEBP2b, polyoma enhancer-binding protein; PIC, pre-initiation complex; Pol I, RNA polymerase I; Pol II, RNA polymerase II; Pol III, RNA polymerase III; RNP, ribonucleoprotein; SRF, serum response factor; STARS, striated muscle activator of Rho signaling; SV, supervillin; SWI ⁄ SNF, switch ⁄ sucrose nonfermentable complex.

monomer availability Transcriptional regulation,

med-iated by cytoskeletal actin dynamics, can be attributed

to modulation of the subcellular localization of

tran-scriptional regulators by ABPs [5] In addition, some

of the mechanisms by which actin affects transcription

and its regulation depend on molecular interactions of

actin with RNA polymerases and components of the

transcription machinery in the nucleus

The role of actin in transcription and

its regulation

Actin is both a major cytoskeletal component of all

eukaryotic cells and also a constitutent of nuclear

pro-tein complexes Nuclear actin plays a role in many

nuclear functions [6–8] First, nuclear actin is required

for transcription by all three nuclear RNA polymerases

Second, nuclear actin associates with small nuclear

ri-bonucleoproteins (RNPs), which have a major role in

mRNA processing [8,9], and is directly involved in the

nuclear export of RNA and cellular proteins [10,11]

Third, nuclear actin also forms complexes with certain

heterogeneous nuclear ribonucleoproteins (hnRNPs)

that bind to and accompany mRNA from the nucleus

to the cytoplasm [12–14] Fourth, nuclear actin and

actin-related proteins have been found in association

with chromatin-remodeling and histone acetyl

transfer-ase complexes, suggesting a role for actin in chromatin

remodeling [15] Recent investigations suggest that

nuclear actin has a role in gene transcription associated

with three main entities: components of the three RNA

polymerases, ATP-dependent chromatin-remodeling

complexes and RNP particles in the eukaryotic cell

nucleus

Nuclear actin is a constitutive component of all

RNA polymerases

Nuclear actin is required for the transcription of all

three RNA polymerases Specifically, b-actin has been

identified as a component of RNA polymerase II

(Pol II) pre-initiation complexes (PICs) Injection of

anti-actin Ig into the nuclei of salamander oocytes

results in contraction of the lateral loops and the

inhi-bition of transcription [8] Furthermore, Hofmann &

de Lanerolle [16] found that actin is associated with

actively transcribed genes and has an essential role in

the activation of transcription In addition, actin is

required for the initiation of transcription through

par-ticipation in the formation of PICs [17] These

conclu-sions are based on the following data: (a) b-actin

participates directly in Pol II transcription, using only

purified transcription factors [18,19]; (b) nascent RNA

molecules are associated with actin in the nuclear matrix and antibodies to b-actin inhibit the synthesis

of nascent transcripts and Pol II transcription [17,19]; (c) adding actin to a highly purified Pol II fraction stimulates transcription [19]; (d) actin colocalizes with transcription sites in early mouse embryos [4,17]; (e) actin is recruited to the promoter region of tran-scribing genes in vivo [19,20]; (f) antibodies to b-actin inhibit the production of a 15-nucleotide transcript that is a prerequisite for the commitment to elongation [19,21]; (g) actin is a component of pre-mRNP parti-cles, and is incorporated into pre-mRNAs by binding

to a specific subset of RNA-binding proteins [4,22]; and (h) actin is a component of PICs and depletion of actin prevents their formation [19,23] The above evidence suggests that there is a strong and specific interaction between actin and Pol II, and actin partici-pates in Pol II transcription What then is the function

of actin in Pol II transcription? From the above data,

we conclude that: (a) based on chromatin immunopre-cipitation assays results, which show that actin is recruited to genes poised to begin transcribing, it is known that actin is involved in recruiting Pol II to the PIC [19]; (b) decreased actin levels resulting from anti-actin Ig inhibit PIC formation by preventing the bind-ing of TBP to the TATA box, indicatbind-ing that PIC for-mation is required for the association of actin with promoter DNA [19]; (c) antibodies to b-actin prevent PIC formation, suggesting that actin acts as a bridge between the polymerase and other constituents of the PIC [24]; and (d) actin and nuclear myosin 1 (NM1),

an isoform of myosin 1, are involved in transcription elongation [6,25,26] Together, these data suggest that actin is involved in multiple stages of the transcription process

b-Actin also has an important role in RNA poly-merase III (Pol III) transcription [27] First, b-actin is tightly associated with Pol III via direct protein–pro-tein interactions with one or more of the RPC3, RPABC2 and RPABC3 subunits, and constitutes part

of the active Pol III [27] Photochemical cross-linking experiments, performed using a transcription initiation complex, indicated that actin makes complex contact with DNA [28] Second, chromatin immunoprecipita-tion assays identified that b-actin is located at the pro-moter sequences of an actively transcribed U6 gene

in vivo, which suggests that it participates in the tran-scription of Pol III [27,29,30] Upon treatment with methane methylsulfonate, a drug that represses Pol III transcription, the U6 initiation complex and b-actin are largely dissociated from promoter sequences [27,29,31] Notably, there is a much larger decrease in the association between b-actin and the U6 promoter

region when compared with the dissociation of Pol III,

which suggests that b-actin dissociates from the Pol III

complex Third, many experiments have shown that

b-actin is required for Pol III transcription [27,29,32]

The monomeric form of actin is required for Pol III

transcription, suggesting that b-actin is essential for

basal RNA polymerase transcription

Actin and NM1 interact with different components

of the RNA polymerase I (Pol I) machinery, and

together serve as a nucleolar motor involved in the

transcription of ribosomal RNA genes [26,33] Recent

studies have revealed that actin is associated with

rDNA genes, and microinjection of anti-actin Ig into

the nuclei of HeLa cells inhibits pre-rRNA synthesis

in vivo [25,34] The interaction of NM1 with actin in

the initiation complex may trigger a conformational

change that favors the transition of Pol I from the

initiation phase to the elongation phase [25,33] NM1

mutants that lack ATPase activity or actin binding are

not capable of associating with Pol I [17], and their

association with rDNA is greatly impaired Moreover,

the association of actin and NM1 with Pol I is

abol-ished in the presence of ATP and is stabilized by

ADP, further suggesting that nuclear actomyosin

com-plexes act as a molecular motor that facilitates

tran-scription [17] NM1 binds the DNA backbone via its

positively charged tail domain, whereas the head

inter-acts with actin bound to RNA polymerase [4] It has

been suggested that by anchoring NM1 to DNA, and

actin to RNA polymerase, an auxiliary motor is

gener-ated that works in concert with nuclear RNA

poly-merases to drive transcription [23] This suggests that

the cooperative action of actin and myosin in the

nucleus is required for Pol I transcription and reveals

an actomyosin-based mechanism in transcription

Actin serves as components of

chromatin-remodeling complexes

Actin is essential for the function of

chromatin-remod-eling complexes in transcriptional activation Nuclear

actin is an ATPase that cycles between monomeric

(G-actin or b-actin) and polymerized (F-actin) states

[4] Eukaryotic cells have several ATP-dependent

chromatin-remodeling complexes, depending on the

ATPase in the complex, as follows: switch⁄ sucrose

nonfermentable (SWI⁄ SNF) complexes, imitation of

SWI-containing complexes, Mi-2 complexes, histone

acetyltransferase complexes, such as the Nu4A and

TIP60 complexes, and INO80 complexes b-Actin is an

integral component of chromatin-remodeling

com-plexes, such as the BAF, BAP and INO80 comcom-plexes,

as well as Nu4A and TIP60 complexes [24,27,29,35–38]

It is generally accepted that chromatin-remodeling com-plexes contain actin, actin-related proteins and⁄ or ABPs Nuclear actin-related proteins (ARP5–9) are associated with actin in chromatin-remodeling com-plexes of the SWI⁄ SNF family, such as those containing the ATPase subunits INO80 or SWR1 [15,24,39] In the SWI⁄ SNF-like BAF complex, b-actin binds directly to the BRG1 ATPase subunit of BAF and stimulates BRG1 ATPase activity, and this interaction is necessary for binding of the BAF complex to chromatin [27,29,40] Actin binding to BRG1 is required for stable association of the complex and provides a link between the chromatin-remodeling complex and the nuclear matrix [5,41] In the INO80 complex, actin is required for efficient DNA binding, ATPase activity and nucleo-some mobilization, as INO80 complexes lacking actin,

as well as the actin-related proteins, ARP4 and ARP8, are deficient for these activities [15] BAF53 and b-actin have also been identified as subunits of the human TIP60 histone acetyltransferase (HAT) complex, which

is involved in DNA repair and apoptosis, and BAF53 is found in a distinct HAT complex involved in c-myc activation, whereas Act3⁄ ARP4 and actin are compo-nents of the yeast Nu4A HAT complex [38,42] In the yeast Nu4A HAT complex, actin and Act3⁄ ARP4 are essential for the structural integity and activity of the complex [38] The presence of actin in chromatin-remodeling complexes suggests that there is a functional link between actin and regulation of the chromatin structure, and a major function of actin is to act as an allosteric regulator in the remodeling of some macro-molecular assemblies, such as chromatin-remodeling factors or transcription complexes

Actin serves as a component of RNP The hnRNP U, a component of pre-mRNP particles, has been shown to interact directly with actin through

a specific and conserved actin-binding site located in the hnRNP U C-terminus and associate with the phos-phorylated C-terminal domain of Pol II [43] Injection

of a peptide acting as a competitive inhibitor of pro-tein–protein contact involving actin and the hnRNP protein, HRP36, into the salivary glands of Chirono-mus tentans disrupts global Pol II transcription as measured by bromo-UTP incorporation; an effect that

is caused, at least in part, by a decrease in elongation measured by run-on assays [22] A recent study has shown that actin binds directly to C tentans hnRNP, HRP65-2, which is a molecular platform for recruit-ment of the HAT histone H3-specific acetyltransferase p2D10 on active genes Both actin and the pre-mRNP protein, HRP65, are complexed in situ with p2D10,

and disruption of the actin–HRP65 interaction releases

p2D10 from Pol II-transcribing genes, coincident with

reduced H3 acetylation and diminished transcription

[6] HRP65-2 binds directly to p2D10, and the

interac-tion between actin and HRP65-2 is required for p2D10

to associate with the transcribed chromatin [6]

More-over, the association of p2D10, actin and HRP65-2

with chromatin is sensitive to ribonuclease digestion,

which indicates that these proteins are tethered to the

transcribed genes by binding to the nascent transcript

These findings support the idea of a link between

nuclear actin, chromatin remodeling and Pol II

transcription [43,44] Obrdlik et al [13,45] identified

that the HAT, p300⁄ CREB binding protein

(CBP)-associated factor (PCAF), associates with actin and

hnRNP U Moreover, it has been shown that

actin, hnRNP U and PCAF associate with the Ser2⁄

5-and Ser2-phosphorylated Pol II C-terminal domain

hnRNP U and PCAF are present at the promoter and

coding regions of constitutively expressed Pol II genes

and are associated with RNP complexes [13] In

sum-mary, these finding suggest that actin, HRP65-2 and

HAT (p2D10 or PCAF) are assembled into nascent

pre-mRNPs during transcription Based on the

evi-dence, it may be proposed that the actin–HRP65-2–

HAT complex is part of the nascent pre-mRNP, and

can travel along the transcribed gene, allowing HAT

to acetylate histones According to this proposal, the

actin–HRP65-2–HAT complex maintains the

chroma-tin in a transcription-competent conformation This

model is supported by the observation that H3

acetyla-tion is reduced and transcripacetyla-tion is inhibited when the

interaction between actin and HRP65-2 is disrupted

[22] In addition, actin-mediated Pol II transcriptional

control may be sensitive to the different polymeriza-tion states of actin [17] Transcrippolymeriza-tionally competent actin may be present in a monomeric or oligomeric form which is different from the canonical actin fila-ments The polymerization states of actin involved in the initiation or elongation phases are different (Fig 1) [43]

Roles of ABPs in the regulation of muscle-specific gene expression The cytoplasmic dynamics of the actin cytoskeleton have been shown to regulate the subcellular localiza-tion of some transcriplocaliza-tion factors, such as the myocar-din-related transcription factors MRTF-A (also referred to as MAL, MKL1 and BSAC) and MRTF-B (also referred to as MKL2 or MAL16) [46,47], the developmentally regulated PREP2 homeoprotein, and the transcriptional repressor Yin-Yang 1 [48,49] Because actin dynamics are regulated by a number of ABPs, ABPs may play a critical role in the regulation

of transcription and gene expression [50] Studies have established that some ABPs induce the formation of actin filaments by their ability to nucleate actin fila-ment polymerization; other ABPs promote filafila-ment breakdown by a mechanism referred to as severing Still other ABPs cross-link or bundle actin filaments or prevent filament formation by their so-called sequester-ing activity Among the notable transcription factors controlled by ABPs are MRTFs, which associate with serum response factor (SRF) and stimulate SRF-dependent transcription [46,51,52] In addition, actin dynamics are regulated by several signal transduction cascades that converge on ABPs [53]

Actin

Actin

Pol II

TF TF TF TBP

CTD

hnRNP U

Actin

Actin

mRNA processing

CTDP

P

P

Pre-mRNA

Ac

Ac

Actin polymerization

? Activator

hnRNP U

PCAF or P2D10

Pol II

HRP65-2

Fig 1 Model for actin–hnRNP U-mediated control of pol II transcription elongation Actin may modulate several steps in Pol II transcription initiation and elongation, either as a monomer or as a polymer Actin may modulate transcription as a monomeric component of transcription preinitiation, chromatin-remodeling and hnRNP complexes During transcription elongation, actin may be recruited to the elongating transcrip-tion machinery via the hyperphosphorylated C-terminal domain and then to the nascent RNP, where actin in complex with the hnRNP U can facilitate recruitment of PCAF or P2D10 to the active gene Formation of actin filaments in the proximity of the Pol II C-terminal domain may help establish a network of interactions between the various factors necessary for transcription elongation and pre-mRNA processing.

MRTF-A associates with G-actin, is predominantly

localized in the cytoplasm of NIH 3T3 cells in the

absence of serum and accumulates in the nucleus in

response to serum stimulation MRTF-B also

under-goes nuclear translocation in response to serum

stimu-lation, although it is less responsive than MRTF-A

[54] Upon activation of RhoA, actin becomes

poly-merized and releases MRTF-A, which in turn

translo-cates to the nucleus to associate with SRF [46]

Striated muscle activator of Rho signaling (STARS) is

a muscle-specific ABP capable of stimulating

SRF-dependent transcription via a mechanism involving

RhoA activation and actin polymerization [55]

Recently, MRTF-A and -B were shown to serve as a

link between STARS and SRF In NIH 3T3 cells

cotransfected with expression plasmids encoding

MRTFs and STARS, the MRTFs are translocated to

the nucleus in the absence of serum The nuclear

local-ization of myocardin is unchanged in the absence or

presence of STARS [54] Thus, STARS may substitute

for serum stimulation and promote the nuclear

translo-cation of MRTFs with the consequent activation of

SRF-dependent transcription Kuwahara et al [54]

found that coexpression of STARS with a

dominant-negative myocardin mutant, which can inhibit the

transcriptional activities of myocardin and MRTF-A

and -B, can completely block the ability of STARS to

induce SRF-dependent transcription in NIH 3T3,

COS1 and 293T cells However, STARS does not alter

the level of expression of MRTFs These observations

suggest that STARS stimulates SRF-dependent

tran-scription solely by promoting the nuclear translocation

of MRTF-A and -B

The STARS protein contains 375 amino acids, with

the conserved ABD contained within the C-terminal

142 residues [55] The STARS C-terminal deletion

mutant, N233, which cannot bind actin or activate

SRF, fails to induce the nuclear accumulation of

MRTF-A and -B By contrast, the C-terminal 142

amino acids of STARS, which bind actin and stimulate

SRF, induce the nuclear accumulation of MRTFs as

efficiently as full-length STARS STARS N233 fails to

enhance MRTF-dependent activation of

SRF-depen-dent reporters, whereas STARS C142 synergistically

enhances MRTF-mediated transcription to the same

level as full-length STARS [55] These results

demon-strate that the ABD of STARS is both necessary and

sufficient for the nuclear accumulation and

transcrip-tional activation of MRTFs by STARS

The activity of STARS involves actin dynamics

Treatment of NIH 3T3 cells with latrunculin B, which

sequesters actin monomers and prevents

Rho-depen-dent nuclear accumulation of MRTF-A and SRF

activation [46], blocks the nuclear accumulation

of MRTF-A and -B in the presence of STARS Conversely, cytochalasin D, which dimerizes actin, but prevents actin polymerization and activates SRF, strongly induces the nuclear translocation of MRTFs, even in the absence of STARS [54] Consistent with these effects on MRTF nuclear import, latrunculin B significantly blocks the stimulatory effect of STARS

on MRTF-dependent transcription, and cytochala-sin D enhances the activity of MRTFs alone These results indicate that actin dynamics are involved in the STARS-induced nuclear accumulation of MRTFs and transcriptional activation of SRF via MRTFs

MRTF-A was recently reported to interact directly with G-actin [56] Unpolymerized G-actin controls MRTF activity [46], and STARS induces actin poly-merization [55] Kuwahara et al [54] demonstrated that expression of wild-type actin, which increases the amount of G-actin, but does not alter the F-actin⁄ G-actin ratio, reduced the ability of STARS to activate MRTF-dependent transcription Wild-type actin did not significantly alter the activity of MRTF in the absence of STARS The actin mutant that favors F-actin formation and increases the F-actin⁄ G-actin ratio [56] stimulates MRTF activity, even in the absence of STARS, and abolishes further activation of MRTFs by STARS By contrast, the actin mutant that

is unable to polymerize and decreases the F-actin⁄ G-actin ratio inhibits MRTF activity and also reduces the ability of STARS to enhance MRTF activity These results suggest that STARS stimulates MRTF activity by inducing the dissociation of MRTFs from actin via depletion of the G-actin pool

The N-terminal regions of MRTFs contain three RPEL motifs which have been shown to sequester MRTFs in the cytoplasm by association with actin [46,56] Consistent with STARS promoting the nuclear import of MRTFs by displacing them from monomeric G-actin, the RPEL motifs are required for the effects

of STARS on MRTFs MRTFs are cytoplasmic, accu-mulating in the nucleus upon activation of Rho GTPase signaling, which alters interactions between G-actin and the RPEL domain Guettler et al [57] showed that the RPEL domain of MRTF-A binds actin more strongly than the RPEL domain of myocar-din, and that the RPEL motif itself is an actin-binding element RPEL1 and RPEL2 of myocardin bind actin weakly compared with MRTF-A, whereas RPEL3 is

of comparable and low affinity in the two proteins Actin binding by all three motifs is required for MRTF-A regulation The differing behaviors of MRTF-A and myocardin are specified by the RPEL1– RPEL2 unit, whereas RPEL3 can be exchanged

between them It has been proposed that differential

actin occupancy of multiple RPEL motifs regulates

nucleocytoplasmic transport and MRTF-A activity

Because myocardin is insensitive to the effects of

STARS, its target genes are expected to be highly

active, irrespective of the polymerization state of actin

However, STARS would be expected to further

aug-ment the expression of these genes via its actions on

MRTF-A and -B, which are also expressed in cardiac

muscle and which form heterodimers with myocardin

In a yeast two-hybrid screen of a skeletal muscle

cDNA library using STARS as bait, Barrientos et al

[58] identified two novel members of the actin-binding

LIM protein (ABLIM) family, ABLIM-2 and -3, as

STARS-interacting proteins These novel proteins

con-tain four LIM domains and a C-terminal villin

head-piece domain, which mediates actin-binding in several

proteins, such as villin and dematin [59] Both

ABLIM-2 and -3 show high homology with ABLIM-1

ABLIM-1 was originally found in the human retina, as

well as in the sarcomeres of murine cardiac tissue, and

was postulated to regulate actin-dependent signaling

[60] Similarly, ABLIM-2 and -3 are expressed in a

tis-sue-specific pattern ABLIM-2 is highly expressed in

skeletal muscle and at lower levels in brain, spleen and

kidney No significant expression has been detected in

the heart In contrast to ABLIM-2, ABLIM-3 is

pre-dominantly expressed in human heart and brain,

whereas the murine ABLIM-3 homolog displays a

somewhat broader tissue distribution that also includes

lung and liver [58]

Both ABLIM-2 and -3 strongly bind F-actin and

colocalize with actin stress fibers The interaction of

STARS with ABLIM-2 and -3 was confirmed by

coim-munoprecipitation and further supported by the

colo-calization of STARS and ABLIM-2, as detected by

immunofluorescence [58] The complementary

expres-sion patterns of ABLIM-2 and -3 in striated muscle

imply that, in vivo, STARS interacts with ABLIM-2 in

skeletal muscle and ABLIM-3 in cardiac muscle

Consistent with the notion that STARS activates

SRF-dependent transcription via stabilization of the actin

cytoskeleton [54], both ABLIM-2 and -3 modulate

STARS-dependent activation of a luciferase reporter

construct controlled by the SM22 promoter, which

contains two essential SRF-binding sites and is highly

sensitive to STARS activity [58] The data suggest that

ABLIM-2 and -3 stimulate STARS activity ABLIM-2

and -3 enhance STARS-dependent SRF-transcription

in COS cells in a dose-dependent manner [58],

suggest-ing that STARS and ABLIMs both physically interact

and functionally synergize to deliver activating signals

to SRF The data imply that, in striated muscle,

STARS plays a critical role in the MRTF-A nuclear translocation process; STARS promotes the nuclear translocation of MRTFs, and thereby SRF-dependent transcription (Fig 2)

STARS activation of SRF-dependent transcription

is mediated, in part, by a Rho-dependent mechanism, because the Rho inhibitor C3 transferase reduces SRF activation by STARS The ability of the Rho kinase inhibitor, Y-27632, to diminish SRF activation by STARS also suggests that Rho kinase is a downstream effector of STARS [55] The Rho family of GTPases, including the best characterized members, Rho, Rac and Cdc42, serve as molecular switches in the regula-tion of a wide variety of signal transducregula-tion pathways [61,62], in particular, actin polymerization and stress fiber formation [63] RhoA signaling has been shown

to induce the nuclear import of MRTF-A in smooth muscle cells, thereby triggering smooth muscle gene activation [64] It is well-known that actin dynamics and Rho signaling are involved in STARS-induced nuclear translocation and transcriptional activation

of MRTFs, and Rho activity is crucial for actin dynamics Kuwahara et al [54] showed that the dominant-negative RhoA mutant inhibits the nuclear accumulation of MRTFs and the stimulatory effect of STARS on the transcriptional activity of MRTFs Although STARS requires Rho activity to induce actin treadmilling and MRTF nuclear translocation, and the inhibition of Rho activity blocks STARS activity, assays of RhoA activity in STARS-transfected cells did not differ from those in untransfected cells Thus,

Fig 2 Model of the involvement of STARS and ABLIM in actin dynamics and SRF-dependent transcription.

STARS does not appear to function as an upstream

activator of Rho, but requires Rho–actin signaling and

changes in actin dynamics to evoke its stimulatory

effects on MRTFs and SRF activity Taken together,

the small GTPase acts downstream of STARS, and it

seems possible that ABLIM integrates signals from the

small GTPases, Rac and RhoA (via STARS) toward

the actin cytoskeleton

Roles of ABPs in the regulation of

nuclear receptor

Nuclear receptors regulated by ABPs include the

glu-cocorticoid receptor, estrogen receptor, androgen

receptor (AR), thyroid receptor and peroxisome

prolif-erator-activated receptor-c Among these, the AR is

the most widely studied and well-characterized The

AR is a ligand-activated transcription factor that

con-trols the expression of genes involved in functions such

as cell proliferation, cell growth, differentiation and

cell death [65,66] The AR contains an N-terminal

domain harboring activation function 1, a central

DNA-binding domain (DBD) and a C-terminal

ligand-binding domain (LBD) containing activation

func-tion 2 [67–70] Upon binding androgens, the AR LBD

undergoes conformational changes leading to

dissocia-tion from chaperones and translocadissocia-tion to the nucleus

[71–74] AR binding to DNA facilitates the

recruit-ment of general transcriptional machinery and

ancil-lary factors that result in the activation or repression

of specific genes in targeted cells and tissues [75] In

the last decade, an increasing number of proteins have

been proposed to possess AR coactivating or

core-pressing characteristics [76,77] Cofactors facilitate AR

transcription function by histone modifications,

chro-matin remodeling and regulation of the AR N-terminal

domain, and the LBD interaction (N⁄ C interaction)

[78–82] All available data suggest that no single

AR-binding protein completely defines the multiple functions of the AR in controlling cellular growth and differentiation in normal and malignant cells [75] Alternatively, AR pleiotropic activities are probably mediated through its binding to specific functional pro-tein complexes to carry out its broad biological func-tions in mammalian cells More than 200 nuclear receptor coregulators have been identified since the first nuclear receptor coactivator, SRC-1, was isolated

in 1995 [83] Among the nuclear receptor coregulators, ABPs and actin monomers bind to the AR, indicating that they also play an important role in AR-mediated transcription (Fig 3) [5,84] For example, supervillin, a nuclear⁄ cytoplasmic F-actin-bundling protein, is able

to interact with the AR N-terminal domain and DBD– LBD This association is enhanced in the presence of androgens [85] In recent years, ABPs have been shown

to elicit increased activity in regulating AR than was previously thought (Table 1)

Filamin, originally identified as a protein that facili-tates nuclear transport of the AR, interacts with the

AR DBD–LBD in a ligand-independent manner [77,86,87] The absence of filamin hampers androgen-induced AR transactivation In the absence of filamin, the receptor–Hsp90 (Hsp90 is a chaperone protein that plays a key role in the conformational change and transcriptional activity of the AR) complex may remain inactive, anchored to the actin filaments, even

in the presence of steroid and an available nuclear localization sequence on the receptor [87] Filamin may act as a mediator between the receptor and the Hsp90, and control the release of activated receptor after ligand binding in AR cytoplasmic trafficking [87,88] Filamin-A (FLNa) interferes with AR inter-domain interactions and competes with the coactivator transcriptional intermediary factor 2 (TIF2) to specifi-cally downregulate AR function [86] When cleaved at the protease-cleavage site between repeats 15 and 16,

A A

r

RE

R AR

AR HSP

AR AR

ABPs

ABPs

CoactivatorsHAT Actin

AR nuclea translocation

AR N/C interaction Coactivator competition

HDAC chromatin condensation

Actin

Pol II

ABPs

Fig 3 Regulation of androgen receptor

gene transcription by actin-binding proteins.

full-length FLNa releases FLNa(16–24) [86–90] This

naturally occurring C-terminal 100 kDa fragment of

filamin, interacting with the motor protein dynein,

may exert its inhibitory effect by interfering with

inter-actions between the N- and C-terminal domains, and

the coactivator functions of the AR [86,91] Full-length

FLNa is bound to the actin cytoskeleton on the cell

surface and perinuclear areas of the cell via its

N-ter-minal ABD In the absence of ligand, AR is localized

predominantly in the cytoplasm, and its hinge domain

and the LBD are tethered to the C-terminal end of

FLNa [86] FLNa(16–24) colocalizes with liganded AR

to the nucleus In the nucleus, FLNa(16–24) disrupts

interactions between the N- and C-termini of the AR,

and interferes with the binding of the coactivator TIF2

[86,91] There is evidence that interaction between the FXXLF (X = any amino acid) motif of the TAD and the LBD reduces coactivator recruitment and binding

of the LXXLL motif of TIF2 [92] Alternatively, FLNa(16–24) may also directly recruit transcriptional repressors to the target promoter or possess intrinsic histone deacetylase activity to inhibit transcription initiation [86] In addition, the recent report of Rho-regulated PAK6 as an AR hinge-interacting kinase [93] suggests that the FLNa(16–24)–AR hinge complex may serve as an integrator for the many cytoskeletal signaling cascades that converge on the AR

Supervillin (SV) was initially identified from blood cells as an ABP and was found to be expressed in skeletal muscles and several cancer cell lines [94]

Table 1 Role of nuclear actin-binding proteins interacting with the androgen receptor AR, androgen receptor; LBD, ligand-binding domain.

Actin-binding

protein

Targeting

sequence Classes Role in the cytoplasm AR effect Mechanism

Direct or indirect association with the AR Region Gelsolin ( )) Actin filament

severing and capping protein

Involved in gel-to-sol transformations;

severs and caps polymeric actin filaments; acts in the actin-scavenging system; inhibits actin polymerization

Coactivator Promotes AR

activity in a ligand-enhanced manner

Flightless I NLS

Actin-remodeling proteins

Possess F-actin-serving activity

Coactivator Does not enhance

the activity of ARs alone, but requires the presence of a p160 coactivator

Direct

a-actinin-2 ( )) Bundling

proteins

Functions as scaffolds for signaling intermediates that stimulate actin elongation; binding partners for ICAM-1

Supervillin NLS F-actin- and

membrane-associated scaffolding protein

Regulates cell-substrate adhesion; organization

of muscle co-stameres;

stimulus-mediated contractility of smooth muscle and myogenic differentiation

Coactivator Increases interaction

frequency with the AR

C-Terminal

proteins

Cytoplasmic transport;

membrane integrity;

cellular adhesion

Coactivator AR cytoplasmic

trafficking

Filamin A NLS? Cross-linking

proteins

Cross-links actin filaments;

recruits F-actin into extended networks

Corepressor Inhibits N ⁄ C,

suppresses TIF2 activation

Transgelin ( )) Cross-linking

proteins

Organizes actin filaments into dense meshworks

SV is localized to the plasma membrane at sites of

intracellular contact The nuclear localization signal is

located in the middle of this protein [95] At low

den-sity, SV shows a punctate distribution localized to the

cytoplasm and nucleus, whereas at high density, SV is

localized almost exclusively to the plasma membrane

SV has been identified as an AR-interacting protein,

which can interact with both N-terminal activation

function-1 and C-terminal activation function-2 of the

AR and plays a role in AR dimerization [85] The

functional coregulator domain of SV is located at

amino acids 831–1281 of bovine origin, which has

putative actin-binding sites and nuclear localization

signals (NLS) [96] Ting et al [96] showed that SV

(amino acids 831–1281) has a better enhancing effect

on AR transactivation than full-length SV and SV

(amino acids 1010–1792) It is possible that by

remain-ing within the nucleus, SV may increase the interaction

frequency with the AR, resulting in a change in AR

conformation to an activated form to facilitate binding

of the androgen response element located in the target

genes SV is relatively weak in promoting

non-andro-genic steroid-mediated AR transactivation, but is

capa-ble of coordinating with other coregulators, including

ARA55 and ARA70, to enhance AR transactivation

[96,97] These results suggest that the final AR activity

may involve balancing and coordinating multiple

coregulators in any given cell In addition, previous

experiments reported that actin and SV potentiate each

other in promoting AR activity [96] Because several

putative actin-binding sites and functional NLS of SV

are important for the AR transactivation function, and

the minimal functional fragment of SV, which only

contains one actin-binding site, is located in the

nucleus, recruiting actin into the chromatin-remodeling

complex is a potential mechanism of co-regulator

activity [96] The actin chelator, latrunculin B, which

attenuates the coregulator function of both full-length

SV and the minimal functional fragment, also identifies

this potential mechanism Furthermore, Rac signaling

stimulates membrane ruffling that further attenuates

the coregulator activity of SV There are two possible

explanations for this: (a) the accumulation of SV in

the membrane prevents it from associating with AR;

and (b) a decrease in the amount of actin monomer

affects SV coregulator activity, which requires actin

monomers [96] However, SV has no effect on the

cytoplasmic–nuclear translocation of the AR, and does

not affect the half-life of the AR [85]

Gelsolin is a multifunctional ABP, implicated in cell

signaling, cell motility, apoptosis and carcinogenesis

[98,99] Gelsolin regulates actin polymerization and

depolymerization by sequestering actin monomers, and

can sever and cap actin filaments [1] Nishimura et al [100] identified gelsolin as an AR-interacting protein that can enhance its transactivation in prostate cancer cells Because gelsolin lacks a nuclear localization sig-nal, it may be cotranslocated into the nucleus upon binding to other proteins [100] Like filamin, gelsolin is able to interact with AR at the time of its nuclear localization to facilitate the nuclear translocation of

AR [87] Increased expression of gelsolin can enhance

AR activity under hydroxyflutamide (HF) with low levels of androgen treatment to maintain AR-mediated growth and theh survival of tumor cells Gelsolin itself interacts with AR LBD via FXXFF and FXXMF motifs and enhances its activity in the presence of androgen The interaction between the N- and C-ter-mini of the AR does not affect gelsolin FXXFF bind-ing to AR LBD, indicatbind-ing that the gelsolin FXXFF motif has a higher affinity for AR LBD [71] Two pep-tides, D1 (amino acids 551–600) and H1–2 (amino acids 665–695) located within AR DBD and LBD, respectively, can block gelsolin-enhanced AR activity [100] Altogether, gelsolin interacts with the AR during nuclear translocation and enhances ligand-dependent

AR activity

Transgelin, also termed SM22a, was first isolated from chicken gizzard as a transformation- and shape change-sensitive ABP [101] Recently, Yang et al [102] characterized transgelin as a potential suppressor of prostate cancer via inhibition of ARA54-enhanced AR transactivation ARA54, a RING finger protein, inter-acts with AR and enhances its transcriptional activity

in a ligand-inducible manner Transgelin does not inter-act directly with the AR, but exerts its effects through recruitment to ARA54 ARA54 can interact with transgelin both in vitro and in vivo in an androgen-inde-pendent manner [102] The data suggest that transgelin might need the specific interaction with ARA54 to sup-press AR transactivation By contrast, transgelin shows little interaction with the AR, ARA70, ARA55, SRC-1, supervillin, gelsolin and CREB binding protein (CBP) Silencing of endogenous ARA54 via its siRNA can abolish the suppressive effect of transgelin on AR function [102] This suggests that transgelin may be able to suppress ARA54-enhanced AR transactivation

by interrupting the interaction between the AR and ARA54, as well as ARA54 homodimerization, resulting

in enhanced cytoplasmic retention and impaired nuclear translocation of ARA54 and the AR

Flightless-1 (Fli-I) is an ABP that can be either asso-ciated with the cytoskeleton or found in the nucleus, but its exact physiologic functions have not been eluci-dated [103] Fli-I can associate directly with the AR and function in cooperation with specific combinations of

other AR coactivators to enhance the ability of the AR

to activate the transcription of AR-regulated genes [77]

Because Fli-I does not enhance AR activity by itself,

but requires the presence of a p160 coactivator, binding

of Fli-I to the AR is apparently insufficient for Fli-I

coactivator function [104] The contacts between Fli-I

and multiple components in the transcription complex

(AR, glucocorticoid receptor-interacting protein 1,

GRIP1, p160 and coactivator-associated arginine

meth-yltransferase 1, CARM1) may result in more efficient

recruitment of Fli-I to the promoter, a more stable

coactivator complex or a more highly functional

con-formation of the coactivator complex Fli-I is a

second-ary coactivator in AR transcription activation [104]

a-Actinin-2 is a major structural component of

sar-comeric Z-lines in skeletal muscle, where they function

to anchor actin-containing thin filaments in a

constitu-tive manner [105] a-Actinin-2 enhances the

transacti-vation activity of SRC-2 and serves as a primary

coactivator for the AR, acting in synergy with SRC-2

to increase AR transactivation function [106] Huang

et al [106] indicated that wild-type a-actinin-2

(con-taining a LXXLL motif) and mutant a-actinin-2

(mutation of the LXXLL motif to LXXAA) both bind

to the AR, but the mutant form shows much weaker

binding than wild-type a-actinin-2 That is to say, the

LXXLL motif in a-actinin-2 has a major role in the

interaction with the AR However, the LXXLL

motif of a-actinin-2 is dispensable for its primary

coac-tivator role in NR functions, because two truncated

a-actinin-2 fragments (encoding 281–700 and 701–894),

lacking the LXXLL motif, and mutant a-actinin-2

(LXXAA) retain the primary and secondary

coactiva-tor functions of wild-type a-actinin-2 In addition,

a-actinin-2 not only serves as a primary coactivator in

the AR, but also interacts synergistically with GRIP1

and enhances GRIP1-induced AR coactivator

func-tions in the presence of cognate ligands [106]

Further-more, a-actinin-4 also binds to the AR and exhibits

coregulating properties a-Actinin-4 may target the AR

for degradation and⁄ or antagonize AR synthesis upon

the addition of androgen In addition, a-actinin-4

negatively regulates AR-mediated transcription [75]

Roles of ABPs in the regulation of

transcription complexes

More and more experiments have identified that

pro-teins traditionally thought to be strictly cytoplasmic

structural factors can influence gene regulation ABPs

transduced the changes in cell structure that occur

dur-ing morphogenesis to the nucleus, resultdur-ing in changes

in gene expression via either the nuclear shuttling of

transcription factors or the assembly of transcriptional regulatory complexes [107]

ABPs can recruit multiple components to transcrip-tion complexes through different types of interactranscrip-tions Fli-I binds both actin and the actin-like BAF53 (BAF complex 53 kDa subunit, BRG1-associated factor), as well as p160 co-activator [104,108] Fli-I can help to secure the association of an SWI⁄ SNF complex to a p160 coactivator complex Fli-I thus helps to coordi-nate the complementary ATP-dependent nucleosome-remodeling activity of the SWI⁄ SNF complex with the histone acetylating (e.g from CBP and p300) and methylating (e.g from CARM1 and protein arginine methyltransferase 1) activities of the p160 coactivator complex [109] In addition, Fli-I and Fli-I LRR-associ-ated protein 1 (FLAP1) have an important role in reg-ulating transcriptional activation by b-catenin and lymphoid enhancer factor⁄ T-cell factor (LEF1 ⁄ TCF) FLAP1 is a key activator, cooperating synergistically with p300 to enhance LEF1⁄ TCF-mediated transcrip-tion by b-catenin Fli-I negatively regulates the synergy

of FLAP1 and p300 [103] Lee & Stallcup [103] found that Fli-I does not bind well to the p300 KIX domain and does not appear to inhibit FLAP1–p300 binding, suggesting that Fli-I does not interfere with the bind-ing of FLAP1 to p300 Fli-I may exert its negative influence by inhibiting the activity of FLAP1 and other essential factors that bind to Fli-I (Fig 4) It is also possible that Fli-I may recruit negative regulators, such

as histone deacetylases (HDACs), CtBP, Groucho and Chibby, to the b-catenin⁄ LEF1 ⁄ TCF transcription complex Both the leucine-rich repeat (LRR) and gels-olin-like domains of Fli-I are required for the negative

Fig 4 Model of Fil-I participation in transcription regulation Fli-I protein can bind to components of the p160 coactivator complex (p160 and CARM1), which has histone acetylating (CBP ⁄ p300) and methylating (CARM1) activities Fli-I can also bind to actin and the actin-like protein BAF53, both of which are components of the ATP-dependent nucleosome-remodeling complex SWI ⁄ SNF.