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RESEARCH ARTICLE

Phosphorylation of the cytoskeletal protein CAP1 controls itsassociation with cofilin and actin

Guo-Lei Zhou1,2,*,`, Haitao Zhang1,2,*, Huhehasi Wu1, Pooja Ghai1,2 and Jeffrey Field3

ABSTRACT

Cell signaling can control the dynamic balance between filamentous

and monomeric actin by modulating actin regulatory proteins. One

family of actin regulating proteins that controls actin dynamics

comprises cyclase-associated proteins 1 and 2 (CAP1 and 2,

respectively). However, cell signals that regulate CAPs remained

unknown. We mapped phosphorylation sites on mouse CAP1 and

found S307 and S309 to be regulatory sites. We further identified

glycogen synthase kinase 3 as a kinase phosphorylating S309. The

phosphomimetic mutant S307D/S309D lost binding to its partner

cofilin and, when expressed in cells, caused accumulation of actin

stress fibers similar to that in cells with reduced CAP expression. In

contrast, the non-phosphorylatable S307A/S309A mutant showed

drastically increased cofilin binding and reduced binding to actin.

These results suggest that the phosphorylation serves to facilitate

release of cofilin for a subsequent cycle of actin filament severing.

Moreover, our results suggest that S307 and S309 function in

tandem; neither the alterations in binding cofilin and/or actin, nor the

defects in rescuing the phenotype of the enlarged cell size in CAP1

knockdown cells was observed in point mutants of either S307 or

S309. In summary, we identify a novel regulatory mechanism of

CAP1 through phosphorylation.

KEY WORDS: Actin dynamics, Actin cytoskeleton, Kinase, Cell

signaling, Cell migration

INTRODUCTIONThe actin cytoskeleton is essential for many fundamental cell

functions such as morphogenesis, cytokinesis, endocytosis, andcell polarization and migration. The cytoskeleton undergoesdynamic changes by regulating the balance of monomeric actin

(G-actin) and filamentous actin (F-actin), a process called actindynamics. One of the types of actin dynamics is ‘treadmilling’(Paavilainen et al., 2004; Pollard and Cooper, 2009), whereby

actin filaments maintain a constant length but are in a state ofdynamic equilibrium. During treadmilling, monomeric actindepolymerizes from the pointed end (or minus end) becauseit has a lower affinity, but polymerizes onto the barbed end

(or plus end), which has a higher affinity. Energy to maintainthis state comes from the hydrolysis of ATP. Treadmilling can be

exploited by cells to cause rapid changes in dynamics. Actindepolymerizing factor (ADF) proteins, such as cofilin sever‘aged’ actin filaments to create shorter fragments and, hence,

increase the amount of filament pointed ends and consequentdepolymerization of monomeric actin from these ends(Andrianantoandro and Pollard, 2006; Michelot et al., 2007).The monomers then undergo nucleotide exchange, i.e. replacing

the bound ADP with ATP, and are recycled to the barbed end ofthe filament for the next round of actin polymerization (Pollardand Cooper, 2009). Repeated cycles of actin filament turnover

drives actin cytoskeletal rearrangement, and cofilin plays acentral role in driving actin filament turnover (DesMarais et al.,2005; Lappalainen and Drubin, 1997). The activity of cofilin is

controlled by the reversible phosphorylation of S3 (Yang et al.,1998). Thus, by modulating phosphorylation at S3, cell signalingpathways can control the actin cytoskeleton in order to regulatecell motility and chemotaxis; although cofilin regulates actin

dynamics by itself it is assisted by other proteins.

Members of the cyclase-associated protein (CAP) family are

cytoskeletal proteins that interact with cofilin and actin toregulate actin dynamics (Hubberstey and Mottillo, 2002; Ono,2013). Mutation or knockdown of CAPs leads to problems with

the actin cytoskeleton, including aberrant cell morphology,polarity and motility (Bertling et al., 2004; Freeman and Field,2000; Kawamukai et al., 1992; Vojtek et al., 1991; Zhang et al.,

2013). CAP regulation of the actin cytoskeleton is widelyconserved, and defects in S. cerevisiae depleted of the geneencoding CAP are rescued by expression of CAP gene

homologues from even distant species (Kawamukai et al., 1992;Matviw et al., 1992; Zelicof et al., 1993; Zhou et al., 1998). CAPsregulate actin dynamics at multiple levels. They bind andsequester actin monomers to prevent them from polymerizing

(Gerst et al., 1991; Gieselmann and Mann, 1992; Zelicof et al.,1996), which helps to maintain a sufficiently large and readilyaccessible G-actin pool that is essential for rapid actin

cytoskeletal reorganization (Zhou et al., 2014). Studies over thepast decade have revealed that several domains within CAPs bindto actin, and that CAPs also facilitate nucleotide exchange of

ATP onto monomers (Hubberstey and Mottillo, 2002; Ono,2013). CAP1 and CAP2 are the two mammalian CAP isoforms.Of the two, CAP1 has been studied more extensively and itsfunction as a regulator of the actin cytoskeleton and cell

migration has been solidly established (Bertling et al., 2004;Moriyama and Yahara, 2002; Zhang et al., 2013; Zhou et al.,2014). Our work here focuses on CAP1.

CAP homologues have three conserved structural domains, theN-terminal domain, the C-terminal domain and a proline-rich

middle domain (Hubberstey and Mottillo, 2002; Ono, 2013). Allthree domains contribute to actin filament turnover throughinteractions with cofilin, and G- and F-actin (Ono, 2013). In

mammals, the C-terminal domain binds and sequesters G-actin,

1Department of Biological Sciences, Arkansas State University, State University,AR 72467, USA. 2Molecular Biosciences Program, Arkansas State University,State University, AR 72467, USA. 3Department of Pharmacology, University ofPennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.*These authors contributed equally to this work.

`Author for correspondence (gzhou@astate.edu)

Received 7 May 2014; Accepted 30 September 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 5052–5065 doi:10.1242/jcs.156059

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and also catalyzes nucleotide exchange of ATP onto ADP-boundG-actin, whereas in yeast this function is further enhanced by the

Wasp homology 2 (WH2) domain, which is located towards theC-terminus of the middle domain (Chaudhry et al., 2010;Makkonen et al., 2013). Nucleotide recharging on ADP–G-actinin complex with cofilin is a key rate-limiting step (Nishida, 1985)

and CAPs relieve the inhibitory effect of cofilin on recharging.The N-terminal domain of CAP binds the cofilin–ADP–G-actincomplex first for subsequent nucleotide exchange, and CAPs can

also directly bind F-actin to promote its severing (Chaudhry et al.,2013; Normoyle and Brieher, 2012). Moreover, the proline-richmiddle domain interacts with profilin (Bertling et al., 2007;

Makkonen et al., 2013). Finally, CAPs were recently shown toassemble into a hexameric oligomer (Chaudhry et al., 2013).Regions within the N-terminal domain (Quintero-Monzon et al.,

2009; Yusof et al., 2005; Yusof et al., 2006) and C-terminaldomain (Dodatko et al., 2004; Zelicof et al., 1996) can mediateCAP assembly into oligomers. However, the regulatory signalsthat modulate CAPs, if there are any, remain unknown.

We report here that mouse CAP1 is a phosphorylatable protein,with at least two phosphorylatable regulatory (hereafter referredto as phospho-regulatory) sites. Phosphorylation at S307/S309

prevents the association with cofilin and expression of mutantsthat fail to undergo this phospho-regulation disrupts the actincytoskeleton. We identify glycogen synthase kinase 3 (GSK3) as

a kinase that phosphorylates residue S309 and suggest that S309functions with S307 as a tandem phospho-regulatory site tocontrol association with cofilin and actin. Thus, phosphorylation

of S309 by GSK3 (and potentially other kinases) is part of aregulatory mechanism that might facilitate association anddissociation of CAP1 with partner proteins cofilin and actin,essential interactions to promote actin filament turnover.

RESULTSMapping of phosphorylation sites on CAP1We first tested potential phosphorylation of CAP1 in vivo throughmetabolic labeling of cells with radiolabeled orthophosphate, aspreviously described (Woodring et al., 2004) followed by

immunoprecipitation of CAP1 from cell lysates with an anti-CAP1

monoclonal antibody (Freeman and Field, 2000). Radioactivitywas detected in CAP1, suggesting that CAP1 is, indeed,

phosphorylatable (data not shown). We next mappedphosphorylation sites on mouse CAP1 by mass spectrometryassays. To do this, we transiently expressed 66His-CAP1 inHEK293T cells (Zhang et al., 2013). 66His-CAP1 in the lysate was

precipitated by using Ni-NTA beads and the 66His-CAP1 bandwas excised from a Coomassie-Blue-stained protein gel andsubjected to mass spectrometry. As shown in Table 1, a total of

nine phosphorylation sites were identified in three independentassays. Some of these sites, such as S36, S307, S309 and T314,were well-conserved among the mammalian CAP1 homologues.

The NCBI ID numbers of the CAP homologues compared by us areas following: (1) S. pombe Cap1: NP_587817.1; (2) M. musculus

CAP1: NP_031624.2; (3) H. sapiens CAP1: NP_001099000.1; (4)

S. cerevisiae CAP (SRV2): NP_014261.1; (5) R. norvegicus CAP1:NP_071778.2; (6) C.elegans CAS-2: NP_491324.1; (7) D.

melanogaster Capt: AAF51408.2; (8) A. thaliana CAP1:NP_195175.1; (9) M. musculus CAP2: NP_080332.1.

S36 and S307/S309 are phospho-regulatory sitesTo assess the significance of each phosphorylation site, we

generated CAP1 point mutants, expressed the mutants in NIH3T3cells and then evaluated their effects on the actin cytoskeleton.The phosphorylation sites were mutated to either alanine (A),

which mimics a non-phosphorylatable residue, or aspartic acid(D), which is phosphomimetic. Where two phosphorylation sitesare adjacent to each other (T24/S25) or are separated by a single

residue (S307/S309), we mutated both sites together. As a result,we generated a total of fourteen constructs that express CAP1mutants fused to green fluorescent protein (GFP), as listed insupplementary material Table S1. These mutants were: T24A/

S25A, T24D/S25D, S36A, S36D, S217A, S217D, S227A, S227D,S247A, S247D, S307A/S309A (hereafter referred to as AA),S307D/S309D (hereafter referred to as DD), T314A and T314D.

We next stably expressed the mutants in NIH3T3 cells andcompared them with wild-type CAP1 (WTCAP1). Comparableexpression levels of the exogenous proteins were verified by

western blotting with a GFP antibody (Fig. 1A, shown only for

Table 1. Phosphorylation sites identified on mouse CAP1 Amino acid residue Conservation among CAP homologues Predicted potential kinases

T24 3/9S25 3/9S36 5/9 ATM, CK1, PRKDC, GRKsS217 5/9S227 3/9S247 3/9S307 3/9 CDKs, GSK3, MAPKs, CDC2, PAKsS309 7/9 CDKs, GSK3, MAPKs T314 5/9 CK2, GSK3, CDC2, PKCs

A. thaliana CAP1 36 SSAAGIDIASSDP 306 —-TSKPAFSKTGPPS. cerevisiae Srv2 64 SAENAPEVEQDPK 358 --PKKPSTLKTKRPS. pombe CAP1 92 SLTSTSAVEAVPA 384 --PSKPAETAPVKPC. elegans CAS2 27 --KKETPVDATPQ 289 --VSENKKPEKIHED. melanogaster capt 1 ------------M 257 --AVAAPSAAQAKAM. musculus CAP2 32 CGEVNGVIAGVAP 311 --TSPKSYPSQKHAR. norvegicus CAP1 32 ADS—-PSKAGAAP 306 TSPSPKPATKKEPAH. sapiens CAP1 32 GDS--PSK-GAVP 307 TSPSPKRATKKEPAM. musculus CAP1 32 GDS--PSK-GAVP 306 TSPSPKPATKKEPA

Numbers to the left of each sequence represent that of the first amino acid in that particular homologue. Residues shown in bold are conserved in the aligned homologues.

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WTCAP1 and S36A, S36D, AA and DD – the four mutants

characterized further). Using phase-contrast microscopy, wefound that expression of WTCAP1 caused increased peripheralruffles (data not shown). In contrast, mutation of two

phosphorylation sites, S36 and S307 and/or S309 causedsignificant, yet distinct, morphological changes. Many cells thatexpressed the S36D mutant were smaller, with multiple randomly

positioned lamellipodia. The cells expressing the DD mutant werelarger and had reduced peripheral ruffling. In contrast, AA-expressing cells – albeit also larger – still had well-developed

lamellipodia. We also found that cells expressing T314A had aphenotype similar to that of AA-expressing cells. To quantify thechanges, we measured the cell area of 50 cells from three fields;results from three independent experiments were analyzed using

Student’s t-test as shown in Fig. 1B. Consistently, S36D-expressing cells were significantly reduced in size, whereasAA-, DD- and T314-expressing cells were significantly increased

in size when compared to cells expressing WTCAP1. We alsoscored cells with prominent peripheral ruffling (lamellipodia) andfound that the DD- and S36A-expressing cells had the least

number of cells with lamelipodia (Fig. 1C). These data suggestthat CAP1 is regulated by phosphorylation within two regions,the first is near residue S36 and the second is within amino acid

residues 307–314. The rest of the mutants did not cause anyobvious morphological changes compared to WTCAP1,suggesting that their phosphorylation has a less significant rolein the regulation of CAP1 function.

Expression of S36 or S307/S309 mutants in NIH3T3 cellsresults in changes of the actin cytoskeletonWe next stained cells with fluorescently tagged (Alexa-Fluor-488- or Alexa-Fluor-594-conjugated) phalloidin to visualize F-actin. When we examined cells cultured on Nunc chamber slides

for wide-field fluorescence imaging, we found that control cells

expressing the GFP vector rarely developed lamellipodia but had

prominent stress fibers (Fig. 2A, upper left panel, cells indicatedby arrows). Expression of WTCAP1 did yield lamellipodia(Fig. 2A, upper right panel, cells indicated by arrows), a

characteristic of motile cells because these structures helpestablish cell polarity and are required for cell migration.Interestingly, many S36D-expressing cells had a disrupted actin

cytoskeleton that appeared collapsed, some of them hadlamellipodium-like protrusions on the ‘sides’ of otherwisespindle-shaped cells (Fig. 2A, middle right panel, arrows

indicate cells with a contracted actin cytoskeleton andarrowheads indicate randomly positioned protrusions). Both theDD- and AA-expressing cells had enhanced stress fibers(Fig. 2A, bottom panels, cells indicated by arrows), and looked

similar to the vector-expressing control cells. The stress fibers inDD- and AA-expressing cells are also similar to those observed inCAP1 knockdown fibroblasts and HeLa cells (Bertling et al.,

2004; Zhang et al., 2013), presumably because of reduceddepolymerization of actin filaments due to impaired CAP1activity. However, AA-expressing cells had more lamellipodia

than DD-expressing cells. We calculated the percentage of cellswith prominent lamellipodia in a way similar to the one describedfor Fig. 1D, and found that S36D- and AA-expressing cells had

similarly high percentages of cells with developed lamellipodia asWTCAP1-expressing cells (Fig. 2B). The changes in cellmorphology and the actin cytoskeleton caused by the mutantsare briefly summarized in supplementary material Table S1.

We further examined the actin cytoskeleton by conductingconfocal microscopy. In addition to enhanced lamellipodia, cellsexpressing WTCAP1 had remarkably reduced stress fibers

(Fig. 2C, right top panel indicated by arrowheads and arrows).The mutants also affected stress fibers in some of the S36D-expressing cells where the stress fibers had a discontinuous

structure (Fig. 2C, right middle panel, indicated by arrows), in

Fig. 1. Expression of phosphomutants of S36 and S307/S309 inNIH3T3 fibroblasts causedmorphological changes.(A) Expression of GFP-fusion WTCAP1and mutants. Cell lysates from theNIH3T3 stable cells harboring an emptyGFP vector, a plasmid expressing GFP-fusion WTCAP1 and the four mutantswere resolved on 10% SDS-PAGEfollowed by western blotting using a GFPantibody. GAPDH was used as a loadingcontrol. (B) Effects of phospho mutantson cell size. The area of 50 cells per fieldwere measured individually (usingImageJ software) from three fields foreach experiment. Results from threeindependent experiments were analyzedby using Student’s t-test, the error barsrepresent 6s.e.m; *P,0.05, **P,0.01.(C) Mutations S36A or S307D/S309Dreduced the percentage of cells withlamellipodia (LP). Cells with lamellipodiawere counted and the percentages werecalculated. 100 cells per field werecounted from three fields for each celltype, and results from three independentexperiments were analyzed and shownas in B.

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Fig. 2. Actin cytoskeletal alterationscaused by expression of CAP1phospho mutants. (A) Wide-fieldfluorescence imaging shows alterations inlamellipodia and stress fibers in cells thatexpress the mutants. The actincytoskeleton was stained withfluorescently tagged phalloidin (Alexa-Fluor 594). Cells indicated by arrows showthe typical changes to the actincytoskeleton, as discussed for S36D- andS307/S309-mutant-expressing cells;arrowheads indicate randomly positionedprotrusions in the S36D-expressing cells.(B) Percentage of cells with prominentlamellipodia. Fifty cells were counted foreach cell type and the experiment wasrepeated for three times. The data wereanalyzed using Student’s t-test and shownas mean 6 s.d; **P ,0.01). (C) Confocalfluorescence microscopy showing furtherdetails of the actin cytoskeletal alterations.F-actin was stained with fluorescentlytagged phalloidin (Alexa-Fluor 488) andimages were taken using a 606lenses witha BD Pathway 855 imaging system (GFPis stably expressed at low levels and doesnot interfere with Alexa-Flour 488 inmicroscopy). Arrows indicate changes tothe stress fibers of each cell type, asdiscussed; arrowheads indicatelamellipodia. (D) Transwell migrationassays revealed reduced motility in bothS36D and DD cells. The data wereanalyzed and are shown as in B; *P,0.05,** P,0.01. The experiment was repeatedthree times with similar results.

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contrast to the straight stress fibers seen in control cells as well asstress fibers typical seen in mammalian cells (not shown). We

speculate that the abnormal stress fiber structure contributes tothe contracted actin cytoskeleton in these cells. Also, weconfirmed significantly enhanced stress fibers in DD- and AA-expressing cells that were also somewhat thicker than those in

GFP-vector-expressing cells (Fig. 2C, bottom panels, indicatedby arrows). Thick stress fibers are known to hinder cell migration(Tojkander et al., 2012). Stress fibers in AA-expressing cells were

similar to those of DD-expressing cells; however, unlike DD-expressing cells, AA-expressing cells still developed lamellipodia(Fig.2C, bottom left panel, arrowheads).

The changes in lamellipodia and stress fibers of S36D- andDD-expressing cells suggest that expression of these mutantsaffects cell migration, such as observed previously in Akt1-

knockout mouse embryonic fibroblasts (MEFs) cells (Zhou et al.,2006). Because changes to lamellipodia and stress fibers weremost pronounced in S36D- and DD-expressing cells, we testedthe motility of these cells in Transwell migration assays. As

shown in Fig. 2D, expression of WTCAP1 stimulated cellmigration when compared to cells expressing the GFP vector.In contrast, expression of either S36D or DD significantly

reduced migration. These results suggest that the randomlyoriented protrusions in S36D cells caused difficulty inestablishing and maintaining cell polarity during directional cell

migration. In addition, it is also possible that the discontinuousstress fibers hindered cell migration, because stress fibersgenerate and transduce tension force to pull up the trailing edge

of the cell during migration. As for DD-expressing cells, they hadthe lowest rate of migration, probably because of defects in theirstress fibers and cell polarity.

Effects of S307/S309 phosphorylation on association of CAP1with cofilin and actin, and evidence that the two residuesfunction in tandemChanges to the actin cytoskeleton caused by the S307/S309mutations suggest an impaired activity of CAP1, which led us toreason that phosphorylation on these sites may affect the association

of CAP1 with cofilin or actin, two binding partners of CAP1. Toavoid the complications of interpreting results caused by thepresence of endogenous CAP1, we used CAP1-knockdown HeLacells to re-express WTCAP1 or CAP1 mutants (Zhang et al., 2013).

Plasmids expressing WTCAP1 or CAP1 mutants were modified byintroducing mismatches to the small hairpin RNA (shRNA)-targetsequence so that they were not recognized by shRNA used to stably

knock down CAP1, but without changing the amino acid sequence.Stable re-expression of 66His-tagged WTCAP1, S36A, S36D, AAor DD mutants was confirmed by western blotting and

immunofluorescence (Fig. 3 and Fig. 5A). We tested cofilinbinding in a GST–cofilin pull-down assay that we had developedpreviously (Zhang et al., 2013). Interestingly, we found striking

differences between the DD and AA mutants in their capabilities tobind cofilin; the DD mutant virtually lost cofilin binding, whereasthe AA mutant had drastically increased cofilin binding that is muchhigher than that of WTCAP1 (Figs 3A, quantified results shown in

Fig. 3B). We also found that the S36A mutant, similar to the DDmutant, had reduced cofilin binding. It is noteworthy that NIH3T3cells expressing the S36A or DD mutant had the lowest number of

lamellipodia (Fig. 1D); these results suggest that cofilin binding iscrucial for CAP1 activity in facilitating the formation of these F-actin-rich structures, and that cofilin binding is regulated by

phosphorylation of S36 and S307/S309.

We next examined actin binding in lysates of S36A-, AA- orDD-expressing HeLa cells by using Ni-NTA beads to precipitate

66His-CAP1, followed by western blotting to detect co-precipitated actin (Zhang et al., 2013). We found that, althoughthe DD mutation did not affect actin binding, the AA mutationhad significantly reduced actin binding (Fig. 3C, quantified

results shown in Fig. 3D). Thus phosphorylation can modulateactin binding as well.

Because AA and/or DD mutants harbor mutations at both S307

and S309, we constructed point mutations at either S307 or S309to determine whether one mutation alone affected cofilin or actinbinding. As shown in Fig. 3E and quantified in Fig. 3F, neither

S307A nor S309A showed increased binding to cofilin observedin the AA mutants. Similar results were observed when we testedactin binding. As shown in Fig. 3G and quantified in Fig. 3H,

although the AA mutant had reduced actin-binding capability,single mutants (S307A and S309A) both bound normally to actin.These results suggest that S307 and S309 function in tandem, andphospho-regulation at both residues collaboratively controls the

interaction of CAP1 with cofilin and actin.Although CAP1 binds both G- and F-actin, it has a preference

for G-actin (Ono, 2013). We next tested whether this preference

is changed in the S307/S309 mutants. We treated lysates fromHeLa cells re-expressing WTCAP1 and these mutants withlatrunculin A to depolymerize the F-actin. As shown in

supplementary material Fig. S1, treatment with latrunculin Aincreased actin binding for both WTCAP1 and the AA and DDmutants in a similar fashion, and the AA mutant consistently had

the least actin binding with or without the treatment. These resultssuggest that the S307/S309 mutation of CAP1 does not alter itsbinding preference for G-actin.

GSK3 phosphorylates S309 on CAP1To aid in identifying kinase(s) for the S307/S309 site, we generateda phospho-specific antibody designed to detect phosphorylated

S307 and S309, as described in Materials and Methods. Westernblotting of transiently expressed GFP-fusion WTCAP1, and theAA and DD mutants show that the phospho-specific antibody

recognized GFP-WTCAP1 and endogenous CAP1 but not theGFP-fusion AA or DD mutants (supplementary material Fig. S2A).We further characterized the antibody in peptide blocking assays tosee whether it only recognizes doubly phosphorylated antigen.

As shown in supplementary material Fig. S2B, the doublyphosphorylated peptide (S307p/S309p) completely blockeddetection of the phospho signal on CAP1 in cells, whereas the

S309-phosphorylated peptide (S309p) modestly blocked the signal,suggesting that the antibody also recognizes CAP1 phosphorylatedat S309 alone, although it seems substantially less effective in

blocking the signal. We detected phospho signals on CAP1 from allcells that we tested, including HEK293T, HeLa, NIH3T3 and thepancreatic cell line hTERT-HPNE (Lee et al., 2003) (Figs 4 and 6;

no data shown for NIH3T3). These results support the notion thatS307/S309 is a phospho-regulatory site across different cell (andtissue) types, although mass spectrometry mapping was conductedwith the protein expressed in HEK293T cells.

By using the Net-Phos2.0 program to identify potential kinasesthat may phosphorylate CAP1, the Ser/Thr kinase GSK3 (Kimand Kimmel, 2000; Woodgett, 2001) was predicted to be one of

the kinases to phosphorylate both S307 and S309 (Table 1).Because GSK3 is a regulator of cell polarity and migration (Kimand Kimmel, 2006; Sun et al., 2009; Wu et al., 2011), we further

investigated GSK3. Three commonly used GSK3 inhibitors LiCl

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(Klein and Melton, 1996), 6-BIO (6-bromoindirubin-39-oxime)(Meijer et al., 2003) and SB216763 (Coghlan et al., 2000) wereused in western blots with the phospho-CAP1 antibody.

Treatment of HEK293T cells with all three inhibitors reducedS307/S309 phosphorylation (Fig. 4A, quantified in Fig. 4B). ThePI3 kinase inhibitor LY294002 caused a modest stimulation of

the phosphorylation, consistent with a negative regulation ofGSK3 through PI3K/Akt signaling (Clodfelder-Miller et al.,2005). We found that forskolin, an activator of protein kinase A

(PKA), reduced the phosphorylation, which may result fromindirect inhibition of GSK3 through activated PKA (Fang et al.,2000). We also tested several other inhibitors – including theERK inhibitor U0126, the JNK inhibitor XVI and the ROCK

inhibitor RKI-1447 – and none of them reduced phosphorylation

at S307/S309 (supplementary material Fig. S3A, quantified insupplementary material Fig. S3B). Thus, the inhibitor datasuggest that GSK3 is a kinase targeting S307/S309. We also

directly tested several kinases that regulate the actin cytoskeletonand/or cell migration, including Akt, LIM domain kinase 1 andthe serine/threonine-protein kinase PAK1 in kinase assays, using

purified porcine CAP1 as a substrate; however, none of thesekinases phosphorylated CAP1 (data not shown). We next silencedGSK3b in HeLa cells by using two previously validated lentiviral

shRNA constructs (Yoeli-Lerner et al., 2009). As expected, theconstructs efficiently knocked down GSK3b and also reducedphosphorylation of S307/S309 (Fig. 4C). We further found thatGSK3 co-precipitates with CAP1 in HeLa cells (Fig. 4D) or

with CAP1 purified from bacteria (data not shown), suggesting

Fig. 3. Altered binding of cofilin and actin for the S307/S309 mutants, and evidence that S307 and S309 function in tandem. (A) GST-cofilin pull-downrevealed loss of cofilin binding of the DD mutant, whereas the AA mutant showed drastically increased cofilin binding. Equal amounts of GST-cofilin boundto glutathione beads (,10–20 mg) were added to lysates containing ,400 mg total protein harvested from CAP1 knockdown HeLa cells re-expressing WTCAP1or the mutants. Tagged CAP1 co-precipitated with cofilin was detected with a 66His antibody and GST-cofilin was detected with a cofilin antibody.(B) Quantification of results shown in A. Three independent experiments of GST–cofilin pull-down were measured by densitometry, and the results wereanalyzed and shown in the graph; error bars represent 6s.e.m.; *P,0.05, **P,0.01. (C) Actin co-precipitation assays reveal reduced actin binding for the AAmutant. Cell lysates prepared from CAP1-knockdown HeLa cells re-expressing WTCAP1 or the mutants were incubated with Ni-NTA beads, and the beadswere precipitated and washed with lysis buffer containing 20 mM imidazole. Samples were resolved on SDS-PAGE, and detected with antibodies against actinand 66His in western blotting. (D) Quantification of results shown in C. Three independent experiments were measured by densitometry, results wereanalyzed and are shown in the graph; error bars represent 6s.e.m.; **P,0.01. (E,F) A single S307A or S309 mutation did not mimic the drastic increase in cofilinbinding, the experiments and data analysis were done in a similar manner as described for A and B. (G,H) A single S307A or S309A mutation did not mimicreduced actin binding. Experiments and data analysis were done as described for C and D; **P,0.01. In panels B, D, F and H, the y-axis represents thecapability for cofilin and/or actin binding; the value for WT CAP1 was set at 1, and values for the mutants are relative to those of WT CAP1.

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a physical interaction between CAP1 and GSK3. Takentogether, these results strongly support the conclusion thatGSK3 phosphorylates CAP1 at S307/S309.

To find out whether S307 or S309 is the GSK3 phosphorylationsite, we performed in vitro kinase assays and using synthesizedCAP1 peptides as substrates. GSK3 only phosphorylates

substrates that are primed through prior phosphorylation of a

serine or threonine residue located four or five residues after itsactual phosphorylation site, i.e. [S/T]xxx[S(P)/T(P)] or [S/T]xxxx[S(P)/T(P)], where x can be any amino acid (Cole et al.,

2004; Frame and Cohen, 2001). Because we also mapped T314 asa site of phosphorylation (Table 1), the combination of S309/T314 (separated by four residues), but not S307/T314 (separated

by six residues), fits the motif for a GSK3 site (Cole et al., 2004).

Fig. 4. GSK3 phosphorylates S309 on CAP1. (A) Treatment of HEK293T cells with GSK3 inhibitors LiCl, 6-BIO and SB216763 all reduced CAP1phosphorylation at S307/S309. Cells were treated with the inhibitors for 14 hrs, and cell lysates were prepared for western blotting with both CAP1 and phospho-CAP1 antibodies. (B) Signals from three independent experiments were measured using densitometry and analyzed using Student’s t-test and plotted in thegraph; error bars represent 6s.e.m; *P,0.05, ** P,0.01. (C) Knockdown of GSK3b with lentiviral shRNA in HeLa cells reduced S307/S309 phosphorylation.HEK293FT cells were transfected using Lentiviral Packaging Mix together with the shRNA constructs. Supernatant medium was collected 48 hrs posttransfection and used to infect HeLa cells. The cells were harvested 48 hrs post infection, and lysates were prepared and used in western blotting. (D) CAP1 co-precipitates with GSK3b. Lysates from CAP1-knockdown HeLa cells re-expressing 6xHis-CAP1 or a control vector were incubated with Ni-NTA beads toprecipitate 6xHis-CAP1. The precipitates were resolved on SDS-PAGE and blotted with both 6xHis and GSK3b antibodies. (E) Kinase assays mapping S309 asthe GSK3 site. Peptides harboring an alanine mutation at either S307 or S309 with a phosphorylated or dephosphorylated T314 (a.a. 305–316,QTSPSPKPATKK) were used as substrates in the kinase assays to test their phosphorylation by GSK3b; the GSK3 substrate GSM peptide(RRRPASVPPSPSLSRHSSHQRR; the serine residue shown in bold is phosphorylated) was used as a positive control. Samples were resolved on a 10–20%Tris-Tricine gradient gel (Bio-Rad) and signals were detected by autoradiography. Asterisks indicate quantified signals from the phosphorylated peptide, thearrowhead indicates partially degraded substrate. (F) Kinase assay results supporting T314 as the priming site for S309. A dephosphorylated T314 abolished thephosphorylation by GSK3; also both GSK3a and GSK3b phosphorylated S309. In E and F, numbers below the gels indicate the intensity of the signals in relationto the positive control.

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We, thus, predicted S309 as the GSK3 phosphorylation site, withT314 as its priming site. To test this we synthesized peptides onthe basis of the mouse CAP1 sequence (QTSPSPKPATKK,

amino acid residues 305–316) harboring S307A or S309A

mutations together with a phosphorylated or dephosphorylatedT314 (all indicated in bold). As shown in Fig. 4E, GSK3bphosphorylated the S307/S309/T314(P) peptide, but a single

S309A mutation (S307/A309/T314(P)) was sufficient to abolish

Fig. 5. Effects of single and double mutations ofCAP1 phosphorylaton sites on the subcellularlocalization of CAP1 and the rescue of theenlarged-cell-size phenotype in the CAP1-knockdown HeLa cells. (A) Confocal imagesshowing cells re-expressing a CAP1 mutant (S309A,S309D, AA, DD, S36A or S36D) or WTCAP1. Cellswere fixed with 3.7% formaldehyde and stained withmouse anti-Xpress followed by Alexa-Flour 594 forCAP1 and Alexa-Flour-488-conjugated phalloidin forF-actin. The arrows indicate peripheral localization ofCAP1 mutants (AA and S309A). (B) Quantification ofcell area (measured with ImageJ software) carriedout similarly as done for that shown in Fig. 1C.Results from three independent experiments wereanalyzed using Student’s t-test and plotted in thegraph where the error bars represent6s.e.m; **P,0.01.

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the phosphorylation. In contrast, an S307A mutation (A307/S309/T314P) had no effect on its phosphorylation by GSK3b. A peptidederived from the known GSK3 substrate glycogen synthase 1

from muscle (GYS1) (RRRPASVPPSPSLSRHSSHQRR; theserine residue shown in bold is phosphorylated) was used as apositive control (Ryves et al., 2002). Thus, we mapped S309 as

the actual GSK3 site. We further demonstrated that T314 is thepriming site for S309. Whereas S307/S309/T314(P) wasphosphorylated by GSK3b, the same peptide harboring a non-phosphorylated T314 (S307/S309/T314) was not phosphorylated

by GSK3b (Fig. 4F). Finally, we also demonstrate that GSK3a,the other GSK3 isoform, phosphorylated S309 as well (Fig. 4F).

Although we found that GSK3 can regulate S307/S309, we notethat other cell signals might also regulate this site becausetreating cells with phorbol 12-myristate 13-acetate (PMA)

reduced phosphorylation at S307/S309. Interestingly, when wetested the association between CAP1 and cofilin, we found thatthe PMA treatment increased cofilin binding (supplementary

material Fig. S4). These results further support our conclusionthat dephosphorylated CAP1 has increased binding with cofilin.

Roles of S307 and S309 in CAP1 function and localizationOur biochemical assay results support the notion that S307 andS309 – a GSK3 site – function in tandem to regulate CAP1

Fig. 6. Effects of GSK3 inhibitionon the actin cytoskeleton andCAP1 localization. (A) Treatmentwith 6-BIO reduced S307/S309phosphorylation of CAP1 in hTERT-HPNE cells. (B) GSK3 suppressioncaused accumulation of stress fibers,reduced cell polarity and loss ofCAP1 enrichment at the leading edgeof the cell. Control cells (top panels)had well-established cell polarity andleading edges (indicated by arrows),cells treated with 6-BIO for 5 hrs lostthis polarity (bottom panels; cellsindicated by arrows). Cells werestained with CAP1 antibody andvisualized with an Alex-Fluor 594goat anti-mouse secondary antibodyin addition to phalliodin conjugated toAlexa-Fluor 488. CAP1 is enriched atthe leading edge of the control cells,whereas the cytosolic areasimmediately behind the leadingedges had very weak CAP1 staining(indicated by arrowheads). Incontrast, CAP1 had a diffuselocalization across the cytosol in cellstreated with 6-BIO, including the areamentioned above. (C) GSK3suppression caused relocalization ofboth WTCAP1 and the AA mutant.CAP1 knockdown HeLa cells that re-express WTCAP1, and AA or DDmutants were treated with 6-BIO, andtagged CAP1 was stained with an X-press antibody followed byvisualization using an Alexa-Fluor594 goat anti-mouse secondaryantibody. The arrows indicateperipheral localization of WTCAP1and the AA mutant, and both of whichwere abolished by suppression ofGSK3. (D) GSK3 inhibition led toreduced cofilin activity, includingdownregulation of cofilin as well aselevated cofilin phosphorylation atS3. HeLa cells were treated with5 mM 6-BIO for 5 hrs, and cofilin andS3-phosphorylated cofilin weredetected in western blotting.

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function. To further assess the role for each amino acid residuethat impacts on CAP1 function in cells, we expressed AA, DD,

S309A or S309D mutants in CAP1-knockdown HeLa cells –which were depleted of at least 90% endogenous CAP1 (Zhanget al., 2013). As shown in Fig. 5A and quantified in Fig. 5B,expression of WTCAP1 rescued the phenotype of the enlarged

cell size of the CAP1-depleted cells as previously observed by us(Zhang et al., 2013). In contrast, the AA and DD mutants failed torescue the phenotype of the enlarged cell size (Fig. 5A,B),

consistent with the phenotypes of NIH3T3 cells caused by thesemutants that suggest impaired CAP1 activities. Interestingly, theS309A and S309D mutants were both able to rescue the

phenotype of the enlarged cell size (Fig. 5A,B), consistent withnormal actin and cofilin binding these S309 point mutants(Fig. 3E-H). In fact, mutants S309A and S309D had an even

more pronounced effect on cell size as compared to the effect byWTCAP1, because large cells were not observed in cells rescuedby these mutants. In contrast, in WTCAP1-rescued cells somelarge cells were still observed (data not shown). We also stained

the transfected CAP1 with an X-press antibody and found that theAA mutant localized predominantly to the cell periphery(indicated by arrows in Fig. 5A) much more strongly than

WTCAP1, whereas the DD mutant showed a diffuse localizationacross the cytosol (Fig. 5A). The S309A mutant also localized tothe cell periphery (indicated by arrows), although not as strongly

as the AA mutant. The S309D mutant had a diffuse localizationsimilar to the DD mutant (Fig. 5A). These results suggest thatphosphorylation of S309 affects CAP1 localization, whereas

phosphorylation of both S307 and S309 affects localization aswell as activity. We also tested the rescue capability of the S36Aand S36D mutants, and found that both mutants were able torescue the phenotype of the enlarged cell size of CAP1-depleted

cells (supplementary material Fig. S5).

Inhibition of GSK3 leads to actin cytoskeletal changes andloss of peripheral CAP1 localizationWe next tested effects of GSK3 inhibition on the actincytoskeleton and CAP1 in hTERT-HPNE pancreatic cells,

which have very well-established cell polarity. We firstconfirmed that GSK3 inhibition also reduced CAP1phosphorylation in this cell line (Fig. 6A). In a wound-healingassay, cells treated with vehicle control (Fig. 6B, upper panels)

showed well-established cell polarity (leading edge indicated byarrows). In contrast, cells treated with 6-BIO lost cell polarity(Fig. 6B, lower panels, indicated by arrows). LiCl had a similar

effect (not shown). These cells also had significantly enhancedand well organized stress fibers compared to the control cells.Moreover, treatment with 6-BIO also abolished CAP1 enrichment

at the leading edge of the cell (Fig. 6B). Instead, CAP1 showed adiffuse localization across the cytosol, including the cytosolicarea immediately behind the leading edge, where virtually no

CAP1 staining was observed in the control cells (Fig. 6B, upperpanels, indicated by arrowheads). The stress fiber phenotype isconsistent with those observed in NIH3T3 cells that expressedmutants DD and AA, neither of the mutants was able to be

regulated through reversible phosphorylation. However, thelocalization of CAP1 in GSK3-suppressed cells – in whichCAP1 is expected to be not phosphorylated at S309 – is not

consistent with the cell peripheral localization of the S309A andAA mutants (Fig. 5A). Thus, an unknown localizationmechanism may be involved in these cells, which is

independent of phospho-regulation of CAP1 by GSK3. We

tested this by inhibiting GSK3 with 6-BIO in CAP1-knockdownHeLa cells that re-expressed WTCAP1 and the AA or DD

mutants, and found that treatment abolished the peripherallocalization of both WTCAP1 and the AA mutant (Fig. 6C),supporting this is, indeed, the case. We next looked into potentialeffects suppression of GSK3 might have on cofilin, a binding

partner of CAP1, and found that the treatment with the inhibitorreduced cofilin expression and also elevated levels of cofilinphosphorylated on S3, suggesting loss of cofilin activity

(Fig. 6D). The effect of GSK3 inhibition on cofilin expressionand phosphorylation are consistent with the phenotype ofenhanced stress fibers and loss of cell polarity. Because CAP1

activity depends on cofilin (Moriyama and Yahara, 2002), theeffect of GSK3 on cofilin may be epistatic to its effect on CAPs.

Increased phosphorylation of S307/S309 in cells with a staticactin cytoskeletonTo assess the role that S307/S309 phosphorylation has in regardto CAP1 function within the actin cytoskeleton, we examined the

possible correlation of phosphorylated S307/S309 and actindynamics by comparing the phosphorylation in cells that undergodynamic reorganization of the actin cytoskeleton with

phosphorylation in cells that have a static actin cytoskeleton.For this, one batch of HEK293T cells was cultured in suspension,whereupon their reorganization of the actin cytoskeleton is

substantially reduced. Another batch was cultured on fibronectin-coated plates, on which they undergo dynamic rearrangement ofthe actin cytoskeleton during early and rapid spreading stage.

Interestingly, we found remarkably elevated S307/S309phosphorylation in cells grown in suspension, or when cellswere cultured for extended time periods (e.g. 48 hrs; the leftlane labeled as 0 min; Fig. 7A) and had reached near full

confluence. In contrast, phosphorylation is significantly reducedin cells cultured on fibronectin-coated plates during the spreadingstage. We quantified both CAP1 and phospho-CAP1 signals,

calculated the ratio of phospho-CAP1 to CAP1 (Fig. 7B), andfound CAP1 was significantly reduced in cells during spreadingstage. These results suggest that cells that undergo a dynamic

reorganization of the actin cytoskeleton have reducedphosphorylation. We propose that CAP1 phosphorylated atS307/S309 is inactive, consistent with the observation that thephosphomimetic DD mutant binds poorly to cofilin and is located

throughout the cytosol.

DISCUSSIONWe identify for the first time a phosphorylation-dependentregulatory mechanism of the versatile actin cytoskeletal proteinCAP1. We show that regulation of both phosphorylation sites – at

S307 and S309 – is important for CAP1 function as both play arole in the interaction of CAP1 with cofilin and actin. Single-point mutations at S307 or S309 did not alter binding to cofilin or

actin; thus phosphorylation of S307 (by another kinase togetherwith that of S309 collaboratively regulate CAP1 function. Wefound that GSK3 phosphorylates S309 by using inhibitors,shRNA and in-vitro kinase assays. GSK3 function has primarily

been studied in its role in development and Wnt signaling (Wuand Pan, 2010). However, it also regulates cell polarity andmigration, and GSK3 targets that modulate tubulin can mediate

some of the relevant cell signals. The role for GSK3 in actinregulation is less established, although ARHGAP35 has beenreported to be a GSK3 target (Jiang et al., 2008) and, thus, GSK3

might regulate the actin cytoskeleton through Rho.

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The double mutation of S307D/S309D (mimicking aconstitutively phosphorylated site) prevented cofilin binding,whereas the non-phosphorylatable double mutant S307A/S309A

showed drastically increased cofilin binding, suggesting thatphosphorylation serves as a regulatory mechanism to controlassociation and/or dissociation of cofilin and CAP1. Releasing

cofilin (and actin) from association with CAP1 followingcompletion of nucleotide exchange on G-actin would allowcontinuous turnover of actin filaments by recycling cofilin.

Although the DD mutant lost cofilin binding, it had normal actinbinding. In contrast, although the AA mutant had dramaticallyincreased cofilin binding, its actin binding was reduced. Actinbinding is not necessary for yeast CAP to bind to cofilin and

assist depolymerization or filament severing (Chaudhry et al.,2013), consistent with our results that the S36A and the DDmutants, which lost cofilin association, had the fewest

lamellipodia. Of note, there are two actin binding sites onCAP1, the cofilin–ADP–G-actin binding site at the N-terminusand the G-actin binding/sequestering site at the C-terminus (Ono,

2013). The reduced actin binding in the AA mutant may reflectreduced binding of G-actin at the C-terminal domain, becausebinding to cofilin (which is in a complex with ADP–G-actin thatinitially binds to the N-terminal domain) is drastically increased.

The observation that the DD mutant, which cannot bind to cofilin,also caused changes to lamellipodia that are very similar to those

observed in CAP1-knockdown cells, suggested thatphosphorylation inactivates CAP1. Inactivation of CAP1 by

phosphorylation is also in line with the fact that GSK3 typicallyphosphorylates and inactivates its substrates (Woodgett, 2001).Our results suggest that the DD mutant is acting dominantly. Thiscould be because of (1) the inability to bind the cofilin–ADP–G-

actin to complete the nucleotide exchange or (2) the interferencewith CAPs itself because the functional form of CAP proteinsappear to be a hexameric oligomer. These results support a

universal role for mammalian CAP1 in balancing the ratio ofactin filaments and monomers, a property that cells can use tomodulate cell polarity and migration (Bertling et al., 2004; Zhang

et al., 2013). Moreover, given the important function for CAP1 incontrolling actin dynamics, it would not be surprising if multiplekinases phosphorylate S307 and/or S309 on CAP1 in order to

allow other cellular signals to regulate this protein.Our findings that the AA and DD mutants both caused changes

to the actin cytoskeleton also suggest that phosphorylation does notmerely inactivate CAP1 but, rather, is a step within a regulatory

cycle, in which alternating phosphorylation and dephosphorylationserves to control cycles of association and dissociation of cofilinand actin. The remarkable difference of the subcellular localization

of the AA and DD mutant also support this notion. In this model,phosphorylation at S307/S309 on CAP1 may facilitate the releaseof cofilin so it can be recycled for the next round of severing. Also,

kinase(s) and phosphatase(s) are expected to work in concert toachieve the reversible phosphorylation of CAP1 to drive thecontinuous cycles of actin filament turnover under physiological

conditions, although we have yet to identify any specificphosphatase for these residues.

An unexpected finding of this study is that phosphorylation atS307/S309 regulates cofilin binding because this site is located

near the C-terminus of CAP1, which is distant from the minimalcofilin binding site that – in turn – is closer to the N-terminus. Itthus remains unclear how phosphorylation at S307/S309 can

regulate binding to cofilin. However, we note that the cofilinbinding site has not been clearly defined. Additionally, mutationsat the extreme N-terminal domain of the yeast CAP homologue

(Srv2) can prevent localization and SH3 binding at the P2 site,which is near the equivalent of S307/S309 (Yu et al., 1999).Thus there are other CAP mutations distant from direct bindingsites that can affect binding to partners. We suggest that

phosphorylation can prevent cofilin binding indirectly.Inhibition of GSK3 can cause loss of cell polarity as well as

accumulation of stress fibers. We propose that GSK3 regulates

CAP1 through at least two mechanisms. First, GSK3 (andpotentially other kinases) phosphorylate CAP1 at S309 andpromote CAP1 localization to the cytosol. Second,

phosphorylation at S309 affects protein–protein interactionswith actin and cofilin. Our data suggest that phosphorylationoccurs not exclusively at S309 but is likely to require a ‘priming’

phosphorylation at T314, and requires at least another kinase tophosphorylate S307. The relative contribution of GSK3 inregulating CAP1 may vary in different cells compared to otherkinases. The loss of this phospho-regulation by GSK3 inhibition

is expected to disrupt CAP1 function and actin dynamics.Additionally GSK3 can also, as observed by us, affect actindynamics through other mechanisms, such as cofilin

phosphorylation and downregulation (Fig. 6). We show thatreduced cofilin activity (including reduced expression) could beresponsible for the unexpected CAP1 localization in GSK3-

suppressed cells, overriding the direct effects of GSK3 on CAP1.

Fig. 7. Phosphorylation at S307/S309 is reduced in cells undergoingdynamic actin cytoskeletal reorganization. (A) In HEK293T cells, CAP1phosphorylation at S307/S309 was compared between cells cultured insuspension (SUS) and cells cultured on fibronectin-coated culture dishes(FN). Cells were harvested at the indicated time points and lysates wereprepared for western blotting with antibodies against CAP1 and CAP1phosphorylated at S307/S309 (pCAP1). GAPDH was used as a loadingcontrol. (B) Densitometric quantification of CAP1 and phospho-CAP1 signalsfrom western blots of three independent experiments; ratios of phospho-CAP1 (pCAP1) versus CAP1 were calculated and plotted in the bar graph,error bars represent 6s.e.m. *P,0.05; **P,0.01.

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The results that compare the single point mutations of S309 andS307 with the AA and DD double mutations in biochemical and

cell biological analyses support the notion that the two residuesfunction in tandem. However, it remains to be determined whetherthe differences in binding cofilin and actin, as well as thesubcellular localization of CAP1, are solely responsible for the

cellular phenotype caused by these mutants. The cellular effectsmay involve additional mechanisms, such as effects on nucleotideexchange, of G-actin and CAP1 oligomerization, and studies are

underway to unravel these potential scenarios. In addition to S307/S309, we also identified S36 as another phospho-regulatory site,although we have yet to identify the responsible kinase. Phospho-

regulation may also be employed in fungal CAP, as we previouslyfound that the N-terminal domain of CAP homologues from L.

.edodes and S. pombe both interact with 14-3-3 protein homologues

(Zhou et al., 2000), a family of chaperon proteins that bindphosphoproteins. We did not observe significant alterations in themorphology of cells that express CAP1 harboring mutations at theother phosphorylation sites that we found; however, we cannot

exclude the possibility that they impact on other aspects of CAP1,such as subcellular localization. Some of these sites, such as S227and S247 are within the middle domain, which is involved in CAP

localization in budding yeast (Freeman et al., 1996).In summary, our work reveals a phosphorylation-dependent

regulatory mechanism of CAP1, by identifying a direct

phosphorylation of CAP1 by GSK3 at S309, which, togetherwith phosphorylation of S307, collaboratively regulates CAP1function through association with cofilin and actin in order

to control the actin cytoskeleton. These findings contributemechanistic insights into how cell signaling controls the actincytoskeleton through molecules that regulate actin dynamics.

MATERIALS AND METHODSMiscellaneous reagents, cell culture, transfection andwestern blottingMonoclonal antibodies against human CAP1 has been previously described

(Freeman and Field, 2000). Mouse anti-GAPDH, goat anti-actin and rabbit

anti-GSKb were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Antibodies against GSK3a and phosphorylated cofilin were from Cell

Signaling Technology Inc. (Danvers, MA). Anti-His antibody, Alexa-Fluor-

488-conjugated phalloidin, Alexa-Fluor-594-conjugated phalloidin, Alexa-

Fluor-488 goat anti-mouse IgG (H+L), fibronectin and platelet-derived

growth factor (PDGF) were all from Life Technologies Inc. (Carlsbad, CA).

Cofilin antibody was from the Cytoskeleton Inc. (Denver, CO). The

phospho-specific polyclonal antibody against S307/S309 was developed by

synthesis and injection of the peptides APKPQTS(P)PS(P)PKPA (amino

acid residues 301–313) into rabbits followed by affinity-purification of the

antibody (Genscript Inc., Piscataway, NJ). Lentiviral packaging mix was

from Sigma Aldrich (St Louis, MO). PH797804, LY294002 and MEK

inhibitor U0126 were from Sellekchem (Houston, TX). Forskolin and H-89

were from LC Laboratories (Woburn, MA). 6-BIO was from Biovision Inc.

(Milpitas, CA) and SB216763 was from Cayman Chemical Co. (Ann

Arbor, MI). JNK inhibitor XVI and ROCK inhibitor RKI-1447 were from

EMD Millipore (Billerica, MA). Peptide substrates were synthesized by

Neo-PeptideTM (Cambridge, MA). Fetal bovine serum (FBS) was from

Hyclone Laboratories Inc. (Logan, UT). NIH3T3, HEK293T and HeLa

cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM),

hTERT-HPNE cells were maintained in 75% DMEM supplemented with

25% medium M3 base. Cell transfection was conducted using FuGene 6

from Roche Diagnostics according to the manufacturer’s protocol.

Recombinant active GSK3a and GSK3b, as well as control GSK3

substrate peptide glycogen synthase 1 from muscle (GSM)

[RRRPASVPPSPSLSRHSSHQRR; the serine residue shown in bold is

phosphorylated] were from Millipore (Billerica, MA). Tissue culture dishes

for immunofluorescence were from MatTek Corp. (Ashland, MA) and

Transwell plates were from Corning Inc. (Corning, NY). Western blotting

was conducted following standard procedures, and cell culture, transfection

and preparation of cell lysates were conducted following procedures

previously described (Zhou et al., 2003).

Mapping of phosphorylation sites on CAP1 by mass spectrometryMetabolic labeling of cells with radiolabeled orthophosphate was done as

previously described (Woodring et al., 2004). A plasmid expressing mouse

CAP1 with a 66His-tag at the N-terminus was transfected into HEK293T

cells, and the expressed 66His-tagged CAP1 was precipitated from the cell

lysate with Ni-NTA beads as described (Zhang et al., 2013). The samples

were resolved on SDS-PAGE followed by Coomassie Blue staining of the

gel. The tagged CAP1 band was isolated, reduced with dithiothreitol (DTT)

and trypsin-digested. Part of the recovered sample was used for protein

identification, the remaining sample was loaded onto a TiO2 column for

purification of the phosphorylated peptides (Cantin et al., 2007). The

samples were subjected to a nanospray/Qstar-XL analysis and the data

obtained was searched against the Swiss protein database. The mass

spectrometry and data analysis were performed by the Proteomics Core

Facility at the University of the Pennsylvania Perelman School of Medicine.

Construction of plasmids and establishment of NIH3T3 cellsstably expressing the mutantsThe plasmid that expresses a 66His-tagged mouse CAP1 has been described

previously (Zhang et al., 2013). To express the S36 and S307/S309 mutants

in CAP1 knockdown HeLa cells, DNA fragments of mouse CAP1 mutants

were amplified by using high-fidelity DNA polymerase and sub-cloning

them into the pcDNA4 vector, following similar procedures as described for

the WT CAP1 gene (Zhang et al., 2013). Point mutants of CAP1 yielding

non-phosphorylatable (A) or phosphomimetic (D) amino acid site(s) were

generated by using pEGFP-N1 vector and the primers listed in supplementary

material Table S2. NIH3T3 cells stably expressing WTCAP1 or the point

mutants were generated by transfection of cells, followed by selection with

1000 mg/ml neomycin for 2 weeks and pooling surviving cells.

Phase-contrast imaging and immunofluorescencePhase-contrast imaging and immunofluorescence were conducted as

described (Zhang et al., 2013). The actin cytoskeleton was stained

with fluorescent (Alexa-Fluor-488- or Alexa-Fluor-594-conjugated)

phalloidin. For immunofluorescence of CAP1, cells were blocked with

3% BSA for 1 hr after fixation and permeabilization, followed by

incubation with a CAP1 monoclonal antibody diluted at 1:200 (Freeman

and Field, 2000) or an X-press antibody. Signals were visualized by

incubation with an Alexa-Fluor-594 goat anti-mouse secondary antibody.

The samples were observed and wide-field images were taken using a

Zeiss fluorescence microscope as previously described (Zhou et al.,

2003); wide-field fluorescence and confocal imaging was conducted as

described previously (Zhang et al., 2013).

Measurement of cell area, percentage of cells with lamellipodiaand Transwell migration assaysAreas of NIH3T3 and HeLa cells were measured by using ImageJ

software and analyzed as described previously (Zhang et al., 2013). To

quantify and compare the percentage of cells showing peripheral ruffling,

cells with prominent peripheral ruffling were counted and the percentage

was calculated. 100 cells per field were counted from three fields for each

experiment, and results from three independent experiments were

analyzed using Student’s t-test and plotted in the graph where the error

bars represent +s.e.m. Transwell assays were conducted as previously

described (Zhou et al., 2006). Briefly, sub-confluent stable cells were

serum-starved overnight and ,36104 cells were plated in triplicate into

Transwell inserts with 8 mm pores placed in 12-well plates containing

DMEM supplemented with 10 mg/ml PDGF. Cells were incubated for

,16 hrs before they were stained, and migrated cells were counted.

Kinase assaysKinase assays were conducted similarly as previously described (Zhou

et al., 2006). 20 mg peptide substrate was mixed with 0.2 mg GSK3 (a or

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b) in a 25 ml reaction mixture containing 20 mM non-radiolabeled ATP in

16kinase assay buffer (10 mM MgCl2, 40 mM HEPES pH 7.4).

Reactions were initiated by the addition of 5 mCi c-32P ATP; the

samples were then incubated at 30 C for 30 min and reactions were

terminated by adding 25 ml of 26Tris-Tricine sample buffer (Bio-Rad).

Samples were boiled and resolved on a 10–20% Tris-Tricine gradient gel

(Bio-Rad), and signals were detected by autoradiography.

AcknowledgementsThe authors thank Shoichiro Ono (Emory University, Atlanta, GA), Peter S. Klein(University of Pennsylvania, Philadelphia, PA), Pamela J. Woodring (The SalkInstitute, La Jolla, CA) and Changhui Wang (University of Pennsylvania,Philadelphia, PA) for helpful discussions, Alex Toker (Harvard University,Cambridge, MA) for the gift of GSK3b shRNA constructs and Chao-Xing Yuan atthe Penn Proteomics Core Facility for his excellent technical assistance.

Competing interestsThe authors declare no competing interests.

Author contributionsG.-L.Z conceived, designed and supervised most of the project, and wrote themanuscript. G.-L.Z., H.Z., H.W. and P.G. conducted the experiments andanalyzed the data. J.F. conceived and supervised the project at the early stage,and H.Z. organized the figures and tables.

FundingWork was supported by the Arkansas Biosciences Institute and an InstitutionalDevelopment Award (IDeA) from the NIGMS of the National Institutes of Health[grant number: P20GM103429-12] to G.-L.Z., who is also supported by a pilot grantfrom Arkansas Breast Cancer Research Program and the University of Arkansasfor Medical Sciences Translational Research Institute [CSTA Grant Award numberUL1TR000039]. J.F. is supported by the National Institutes of Health [grant numberR01 GM48241]. Deposited in PMC for release after 12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.156059/-/DC1

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