Redox Modification of Nuclear Actin by
MICAL-2 Regulates SRF Signaling
Mark R. Lundquist,1Andrew J. Storaska,2Ting-Chun Liu,3Scott D. Larsen,4Todd Evans,3Richard R. Neubig,2,5
and Samie R. Jaffrey1,*
1Department of Pharmacology, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA
2Department of Pharmacology, University of Michigan, Ann Arbor, MI, 48109, USA
3Department of Surgery, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA
4Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
5Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA
The serum response factor (SRF) binds to coactiva-
tors, such as myocardin-related transcription factor-
A (MRTF-A), and mediates gene transcription elicited
by diverse signaling pathways. SRF/MRTF-A-depen-
dent gene transcription is activated when nuclear
MRTF-A levels increase, enabling the formation of
transcriptionally active SRF/MRTF-A complexes.
The level of nuclear MRTF-A is regulated by nuclear
G-actin, which binds to MRTF-A and promotes its
nuclear export. However, pathways that regulate nu-
clear actin levels are poorly understood. Here, we
show that MICAL-2, an atypical actin-regulatory pro-
tein, mediates SRF/MRTF-A-dependent gene tran-
scription elicited by nerve growth factor and serum.
increases MRTF-A in the nucleus. Furthermore, we
show that MICAL-2 is a target of CCG-1423, a small
molecule inhibitor of SRF/MRTF-A-dependent tran-
scription that exhibits efficacy in various preclinical
disease models. These data identify redox modifica-
tion of nuclear actin as a regulatory switch that medi-
ates SRF/MRTF-A-dependent gene transcription.
Serum response factor (SRF) mediates gene transcription
induced by serum, various growth factors, and G-protein-
coupled receptor signaling pathways (Posern and Treisman,
2006). SRF-dependent gene transcription is modulated by SRF
coactivators, including ternary complex factor (TCF) and myo-
cardin-related transcription factor A (MRTF-A) (Shaw et al.,
1989; Wang et al., 2002). MRTF-A binds to SRF, forming a com-
plex that influences SRF binding to the CArG box promoter
element, which is found in SRF target genes (Miralles et al.,
2003; Treisman, 1986). SRF/MRTF-A-dependent gene tran-
scription mediates diverse cellular processes including cellular
migration (Leitner et al., 2011), cancer cell metastasis (Brandt
et al., 2009; Medjkane et al., 2009), mammary myoepithelium
development (Li et al., 2006), and neurite formation (Kno ¨ll and
Nordheim, 2009; Wickramasinghe et al., 2008).
SRF/MRTF-A-dependent gene transcription is induced when
MRTF-A localizes to the nucleus (Posern and Treisman, 2006).
MRTF-A is found in both the cytosol and the nucleus but exhibits
increased nuclear localization in response to various signaling
pathways. The nuclear localization of MRTF-A enables it to
form complexes with SRF, resulting in transcription of genes
that contain promoter elements that bind the SRF/MRTF-A
complex (Posern and Treisman, 2006). Thus, SRF/MRTF-A-
dependent gene transcription is highly influenced by the levels
of nuclear MRTF-A.
Recent studies have shown that MRTF-A localization is
regulated by actin dynamics in the nucleus (Baarlink et al.,
2013; Vartiainen et al., 2007). G-actin in the nucleus binds to
MRTF-A, enabling it to be exported to the cytosol (Vartiainen
et al., 2007). Thus, high levels of G-actin in the nucleus seen
during serum deprivation lead to low levels of nuclear MRTF-A.
when signaling pathways reduce nuclear G-actin, which pre-
vents MRTF-A export, resulting in accumulation of MRTF-A in
the nucleus (Vartiainen et al., 2007).
G-actin levels in the nucleus can be regulated by F-actin for-
mation in the cytosol. When actin polymerization is induced in
the cytosol, for example following RhoA-induced stress fiber for-
mation, cellular actin becomes sequestered in cytosolic stress
fibers, leading to the depletion of G-actin throughout the cell
(Vartiainen et al., 2007). RhoA-dependent depletion of G-actin
in the nucleus subsequently activates SRF/MRTF-A-dependent
gene transcription in NIH 3T3 cells (Vartiainen et al., 2007).
The depletion of monomeric actin by cytosolic stress fibers
is unlikely to mediate SRF/MRTF-A signaling in all cell types.
For example, SRF/MRTF-A signaling regulates axon growth (Lu
and Ramanan, 2011) and other neuronal functions (Kno ¨ll and
Nordheim, 2009; Wickramasinghe et al., 2008), but stress fiber
formation is not typically seen in neurons. Therefore, additional
pathways that induce SRF/MRTF-A signaling remain to be
Here, we describe a mechanism that regulates SRF/MRTF-A-
dependent gene expression which involves depolymerization of
Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc. 563
Figure 1. MICAL-2 Is a Nuclear Protein that Depolymerizes Actin
(A) Cherry-MICAL-2 and -3 expressed in HEK293T cells are localized to the nucleus (blue), while Cherry-MICAL-1 and NLS mutant Cherry-MICALs (M2NLSMut
and M3NLSMut) exhibited cytosolic localization. Scale bar, 10 mm.
(legend continued on next page)
564 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
nuclear actin by MICAL-2, a member of a family of recently
described atypical actin-regulatory proteins (Terman et al.,
2002). MICAL-2 is homologous to MICAL-1, an enzyme that
binds to F-actin in the cytosol and triggers its depolymerization
through a redox modification of methionine (Hung et al., 2011;
2010). We show that MICAL-2 is enriched in the nucleus and
induces depolymerization of F-actin in the nucleus. Expression
of MICAL-2 reduces nuclear actin, resulting in nuclear retention
of MRTF-A and subsequent activation of SRF/MRTF-A-depen-
dent gene transcription. We find that MICAL-2 promotes SRF/
MRTF-A-dependent gene expression in several cell types, and
mediates NGF-dependent neurite growth in neuronal cells.
Furthermore, CCG-1423,a small molecule
pathway inhibitor that exhibits efficacy in various preclinical
disease models, directly binds MICAL-2 and inhibits its activity.
Together, these data show that SRF/MRTF-A signaling is regu-
lated by MICAL-2-dependent redox regulation of nuclear actin.
MICAL-2 Is Enriched in the Nucleus
Because of the important roles of MICAL-1 in depolymerizing
actin in axonal growth cones (Hung et al., 2010), we sought to
understand if MICAL-2 and -3 have related functions. In order
to understand the roles of MICAL-2 and -3, we examined the
subcellular localization of each MICAL isoform. Cherry-tagged
MICAL-1 was cytoplasmic in HEK293T cells; however, Cherry-
MICAL-2 and Cherry-MICAL-3 were nuclear (Figure 1A). To
confirm that these localizations are not due to the presence of
the Cherry tag, we monitored the localization of endogenous
MICALs by subcellular fractionation using isoform-specific anti-
bodies (Figures S1A and S1B available online). MICAL-1 was
cytoplasmic, while MICAL-2 and -3 were enriched in the nuclear
fraction in HEK293T cells (Figure 1B). The nuclear localization
of MICAL-2 was also confirmed by immunofluorescence in
HEK293T, COS7, and HeLa cells (Figures S1C and S1D).
The PredictProtein algorithm (Rost et al., 2004) revealed a
putative bipartite nuclear localization sequence (NLS) in both
MICAL-2 and -3, which was not found in MICAL-1 (Figure 1C).
the nuclear localization of MICAL-2 and -3 (Figure 1A), confirm-
ing that these sequences constitute a NLS domain in these
MICAL-2 Redox Activity InducesActin Depolymerization
Because MICAL-2 and -3 share 60% and 63% identity, respec-
domain (Terman et al., 2002), we asked if they also depolymerize
F-actin. To test this, we purified the bacterially expressed cata-
lytic domain and the adjacent actin-binding calponin homology
(CH) domain (designated ‘‘redoxCH’’) for each MICAL isoform
(Hung et al., 2010). To measure actin depolymerization, we
usedapreviouslydescribedassayfor MICAL-1-dependent actin
depolymerization using pyrene-labeled actin (Hung et al., 2010).
As expected, MICAL-1redoxCH-induced actin depolymerization in
an NADPH-dependent manner (Hung et al., 2010) (Figure 1D).
Similarly, MICAL-2redoxCHand -3redoxCH-induced actin depoly-
merization in an NADPH-dependent manner. The rate of NADPH
consumption by MICAL-2 and -3 was markedly enhanced by
F-actin, suggesting that F-actin is a specific substrate of these
enzymes (Figure S1E).
MICAL-2 Regulates Nuclear Actin Polymerization
The ability of MICAL-2 and -3 to depolymerize F-actin in vitro,
combined with their nuclear localization, suggests that these iso-
forms may regulate actin polymerization within the nucleus. We
therefore examined the effect of inhibiting MICAL-2 activity on
F-actin levels within the nucleus in various cell lines. Expression
of either of two dominant-negative MICAL-2 constructs, MICAL-
and MICAL-2GV, which contains a glycine to valine mutation at
amino acid 95 that blocks FAD-binding in flavoprotein monooxy-
actin into linear filaments that span large portions of the nucleus
(Figures 1E and S1F). Similar mutations in MICAL-3 expression
constructs did not appear to alter nuclear actin (data not shown).
MICAL-2 knockdown (Figure S1B) resulted in the appearance
of nuclear F-actin as detected by phalloidin staining in fixed cells
(Figure S1G).Similarly, expression of GFP-tagged LifeAct, which
binds to F-actin (Riedl et al., 2008), revealed nuclear F-actin in
live cells expressing MICAL-2-specific shRNA (Figure S1H).
The MICAL-2 knockdown constructs did not affect cellular
viability (Figures S1I and S1J).
To validate that these actin filaments are localized within the
nucleus, we costained cells with an antibody to lamin A/C, a
marker of the inner nuclear membrane (Figure S1K). Actin fila-
ments were indeed within the nucleus, rather than surrounding
(B) Subcellular fractionation of MICAL isoforms. Endogenous MICAL-2 and -3 are localized to the nucleus while MICAL-1 was detected in the cytosol. MEK1/2,
VDAC, Calnexin, and PCNA immunoblotting was used to confirm the purity of the cytosolic (Cyto), mitochondrial (Mito), microsomal (Micro), and nuclear (Nuc)
(C) MICAL-2 and -3 each contain a putative bipartite NLS. The domain structure of MICAL-2 is indicated above. The enzymatic domain contains a GXGXXG,
aspartate-glycine (DG), and a glycine-aspartate (GD) motif, which is the characteristic FAD-binding motif. The F-actin-binding CH domain and LIM domain
are indicated. A potential bipartite NLS (red box) in MICAL-2 and MICAL-3 is not found in MICAL-1. Shown in blue letters are the amino acids that constitute the
NLS in MICAL-2 and -3. Shown in red are the amino acids that are mutated in the M2NLSMut and M3NLSMut. Uppercase letters are amino acids conserved in
MICAL-1, -2 and -3.
(D) MICAL-2 and -3 depolymerize F-actin similarly to MICAL-1. MICAL-1redoxCH, MICAL-2redoxCHand MICAL-3redoxCHwere incubated with pyrene-labeled actin
and NADPH as indicated. These data indicate that MICAL-2 and MICAL-3, like MICAL-1, induce NADPH-dependent F-actin depolymerization. Statistical sig-
(E) Dominant-negative inhibition of MICAL-2 lead to increased nuclear F-actin. Expression of dominant-negative MICAL2 (MICAL-2CT or MICAL-2GV, green)
resulted in the appearance of filaments (yellow arrows) throughout the nucleus (blue), as seen by phalloidin labeling (red staining, blue arrows). The dominant-
negative constructs colocalized with the F-actin (yellow). Scale bar, 5 mm.
See also Figure S1
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shRNA MICAL-2 #2 shRNA MICAL-2 #1
Nuclear/Cytosolic MRTF-A Ratio
Nuclear/Cytosolic MRTF-A Ratio
Nuclear/Cytosolic MRTF-A Ratio
shRNA MICAL-2 #2
shRNA MICAL-2 #1
shRNA MICAL-2 #1
shRNA MICAL-2 #2
shRNA MICAL-2 #1
shRNA MICAL-2 #2
(legend on next page)
566 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
the nuclear membrane (Figures S1L–S1O). Collectively, these
data indicate that MICAL-2 enzymatic activity inhibits polymeri-
zation of nuclear actin in cells.
MICAL-2 Expression Induces Nuclear Localization of
Since MICAL-2 regulates F-actin within the nucleus, and since
nuclear G-actin mediates nuclear export of MRTF-A (Vartiainen
et al., 2007), we considered the possibility that MICAL-2 reduces
nuclear MRTF-A by promoting its export. To test this, HEK293T
cells were infected with a virus expressing either GFP or
GFP-MICAL-2 and serum-starved for 18 hr under low-serum
conditions (0.3% fetal bovine serum [FBS]) (Miralles et al.,
2003) (Figures 2A and 2B). Contrary to our expectation, overex-
pression of MICAL-2 under these conditions caused a marked
increase in nuclear localization of MRTF-A (Figures 2A and 2B).
We next asked whether the nuclear localization of MRTF-A
induced by serum is mediated by MICAL-2. Treatment of
serum-starved HEK293T cells with 10% FBS for 10 min altered
MICAL-2 phosphorylation levels, suggesting that serum may
regulate the function of MICAL-2 (Figures S2A and S2B). To
determine whether MICAL-2 is required for serum-mediated
accumulation of MRTF-A in the nucleus, we monitored MRTF-A
localization following knockdown of MICAL-2. In HEK293T cells
expressing either of two MICAL-2-specific shRNAs, but not
control shRNA, serum-induced localization of MRTF-A to the
nucleus was markedly reduced (Figures S2C and S2D). Thus,
MICAL-2 induces nuclear localization of MRTF-A in the absence
of serum stimulation and is required for the increase in nuclear
MRTF-A levels after serum stimulation.
We considered the possibility that MICAL-2 could increase
nuclear MRTF-A by increasing expression levels of MRTF-A.
However, MICAL-2 expression has no discernable effect on
global levels of MRTF-A (Figure S2E). Additionally, the nuclear
accumulation of MRTF-A does not appear to reflect binding of
MRTF-A to MICAL-2 (Figures S2F and S2G). Lastly, expression
of MICAL-2 did not induce stress fibers or increase cytosolic
F-actin levels in HEK293T cells (Figures S2I–S2L). Thus,
MICAL-2 does not increase nuclear MRTF-A by increasing cyto-
MICAL-2 Mediates NGF-Dependent Nuclear
Localization of MRTF-A
We next examined whether MICAL-2 is required for nuclear
localization of MRTF-A after growth factor stimulation. MRTF-A
was readily detectable in the cytosol in untreated PC12 cells
and exhibited increased nuclear localization in response to
NGF treatment (Figures 2C and 2D). However, MRTF-A nuclear
localization in response to NGF was markedly reduced by
expression of either of two MICAL-2-specific shRNAs, but not
by a control shRNA (Figures 2C and 2D).
We next asked whether MICAL-2 promotes the nuclear local-
used rat embryonic day (E) 14 dorsal root ganglia (DRG) sensory
neurons, which are typically cultured in NGF. Because these
neurons require NGF for survival (Chun and Patterson, 1977),
we did not test culture conditions lacking NGF. In these neurons,
MRTF-A was readily detectable in both the nucleus and cyto-
plasm (Figures 2E and 2F). Infection of these cells with lentivirus
expressing either of two MICAL-2-specific shRNAs resulted in a
marked decrease in nuclear MRTF-A (Figures 2E and 2F). Taken
together, these data indicate that MICAL-2 is required for effi-
cient nuclear localization of MRTF-A.
MICAL-2 Expression Induces the SRF/MRTF-A-
Because MICAL-2 induces nuclear localization of MRTF-A, we
asked whether MICAL-2 activates SRF/MRTF-A-dependent
gene transcription. To test this, we determined the effect of
MICAL-2 expression on a transcriptional reporter containing
luciferase under the control of a CArG [CC(A/T)6GG] element.
In serum-starved HEK293T cells, minimal levels of luciferase
were detected (Figure 3A). However, expression of MICAL-2
This effect was not seen following expression of MICAL-2
mutants that were catalytically inactive or which contain NLS
mutations (Figure 3A). MICAL-2 did not induce a transcriptional
reporter containing the highly related TCF/ETS SRE element,
which requires SRF, but not MRTF-A (Buchwalter et al., 2004)
(Figure 3B). Collectively, these data indicate that MICAL-2 in-
duces SRF/MRTF-A-dependent reporter gene expression.
MICAL-1 and MICAL-3 overexpression did not activate the
reporter, indicating that activation of SRF/MRTF-A-dependent
gene expression is limited to MICAL-2 in HEK293T cells (Fig-
ure 3C, S3A, and S3B).
To confirm that MICAL-2-mediated activation of SRF/MRTF-A
gene transcription requires MRTF-A, we expressed MRTF-A-
DTAD, a dominant-negative MRTF-A mutant. This protein lacks
the transcription activation domain (TAD) that is necessary for
an active SRF/MRTF-A complex (Wang et al., 2003). Expression
Figure 2. MICAL-2 Induces the Nuclear Localization of MRTF-A
(A) MICAL-2 expression (green) induces nuclear localization of MRTF-A (red) under serum-starvation. Nuclei are outlined in white, based on DAPI staining (blue).
Scale bar, 5 mm.
(B)Quantification of(A).Theaverage intensityof MRTF-Aimmunofluorescence wasquantified inthenucleusand thecytoplasm. Thenucleus:cytoplasmic ratioof
MRTF-A increased 10.76-fold in GFP-MICAL-2-expressing cells compared to GFP-expressing cells. ***p < 0.0005, Student’s t test, n R 30.
(C) MICAL-2 is required for NGF-induced nuclear localization of MRTF-A in PC12 cells. NGF treatment induces nuclear localization of MRTF-A in LacZ shRNA-
to uninfected cells (blue arrows). Nuclear border indicated by dotted white lines. Scale bar, 10 mm.
(D) Quantification of (C). ***p < 0.0005, ANOVA (***p < 0.0001) with Dunnett posttest n R 30. All data in this figure are mean ± SEM.
(E) The nuclear localization of MRTF-A in dissociated E14 DRG neurons cultured in the presence of NGF is dependent upon MICAL-2. Infection of DRG neurons
with MICAL-2-specific shRNA results in a substantial nuclear depletion of MRTF-A (red). Scale bar, 5 mm.
(F) Quantification of (E). ANOVA (***p < 0.0005) with Dunnett posttest. n R 25.
See also Figure S2.
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