Direct Interaction of 14-3-3ζ with Ezrin Promotes Cell Migration by Regulating the Formation of Membrane Ruffle

    Direct interaction of 14-3-3ζ with ezrin promotes cell migration by regulating the formation of membrane ruffle Miaojuan Chen, Tengfei Liu, Lina Xu, Xuejuan Gao, Xiaohui Liu, Cuihua Wang, Qingyu He, Gong Zhang, Langxia Liu PII: DOI: Reference:

S0022-2836(14)00337-4 doi: 10.1016/j.jmb.2014.06.021 YJMBI 64499

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

26 February 2014 26 June 2014 27 June 2014

Please cite this article as: Chen, M., Liu, T., Xu, L., Gao, X., Liu, X., Wang, C., He, Q., Zhang, G. & Liu, L., Direct interaction of 14-3-3ζ with ezrin promotes cell migration by regulating the formation of membrane ruffle, Journal of Molecular Biology (2014), doi: 10.1016/j.jmb.2014.06.021

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ACCEPTED MANUSCRIPT Title: Direct interaction of 14-3-3 with ezrin promotes cell migration by regulating the formation of membrane ruffle

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Authors: Miaojuan Chen#, Tengfei Liu#, Lina Xu, Xuejuan Gao, Xiaohui Liu, Cuihua

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Wang, Qingyu He, Gong Zhang, Langxia Liu*

Affiliation: Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China.

Correspondence to: Prof. Langxia Liu, Institute of Life and Health Engineering,

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Jinan University, Guangzhou 510632, China.

Email: [email protected], [email protected]

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These two authors contributed equally to this work.

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#

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Fax: +86-20-85222573

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Phone: +86-20-85222573

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ACCEPTED MANUSCRIPT Abstract 14-3-3 proteins have been shown to regulate the actin cytoskeleton remodeling, cell

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adhesion and migration. In this study，we identified ezirn, a cross-linker between

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plasma membrane and actin cytoskeleton as a novel 14-3-3ζ interacting partner. The direct interaction between 14-3-3ζ and ezrin was validated in the cells and by in vitro assays. We showed that the 14-3-3ζ binding region in ezrin was located within the

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N-terminal and central α-helical domains, and that the αG to αI helices of 14-3-3ζ are

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responsible for the binding to ezrin. Functional analyses revealed that the regulation of cell migration and membrane ruffling by 14-3-3ζ is ezrin-dependent, for which the

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integrity of ezrin protein was required. Conversely, the knockdown of 14-3-3ζ

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abrogates also the stimulatory effect of ezrin on cell migration and membrane ruffling.

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Moreover, we found that the phosphorylation of Thr567 in ezrin facilitates the 14-3-3ζ-ezrin interaction and the formation of membrane ruffles. Taken together,

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these results suggest strongly that the functions of these two proteins in cell migration are linked, and might be mediated by their direct physical interaction which is important for the formation of membrane ruffles. Key words: Protein-protein interaction; actin cytoskeleton remodeling; cell migration; 14-3-3 zeta; ezrin 2

ACCEPTED MANUSCRIPT Introduction The 14-3-3 proteins are a group of conserved acidic proteins in eukaryotic cells

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that bind phosphoserine/phosphothreonine motifs. Seven 14-3-3 isotypes (α/β, γ, τ/θ, ε,

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η, σ, ζ/δ), encoded by separated genes, are found in mammals [1]. 14-3-3 proteins are universal adaptor proteins involved in the regulation of diverse cellular processes, including cell proliferation, cytoskeleton organization and cell migration, cell cycle

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checkpoint control and apoptotic signaling response. Particularly, 14-3-3 proteins

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have been shown to regulate the actin cytoskeleton remodeling, cellular adhesion and migration through interaction with actin filaments-associated proteins, various kinases,

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small GTPases, and adhesion molecules which play roles in the reorganization of

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actin filaments and cellular adhesion and motility [2].

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The ζ isoform of 14-3-3 is widely expressed in the tissues, and is one of the most studied isoforms. During the past years, it has emerged as a potential prognostic and

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therapeutic target protein, especially owing to its role to promote cell proliferation, adhesion and survival, and to inhibit apoptosis in multiple cancers [3-5]. 14-3-3ζ is overexpressed in many human cancers, including multiple myeloma, breast cancer, lung cancers, esophageal cancer, oral squamous cell carcinomas, stomach cancers, prostate

tumor,

and

papillomavirus

induced

carcinomas

[6-15].

14-3-3ζ

overexpression occurred in 42% of breast cancers [16]. The elevated expression of 14-3-3ζ was correlated with the progression and grade of non-small cell lung cancers (NSCLC), and poor survival rates of the patients [17]. In our previous studies, we have observed that the treatment of human multiple myeloma U266 cells by arsenic 3

ACCEPTED MANUSCRIPT increased significantly 14-3-3ζ expression. In order to investigate the molecular mechanisms of action of 14-3-3ζ in the cell and in the physiopathology of cancer, we

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used co-immunoprecipitation in tandem with LCMS/MS analysis to identify the new

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interacting partners of 14-3-3ζ in human multiple myeloma U266 cells [6]. Among the identified candidates, ezrin, the prototype member of ERM (Ezrin-Radixin-Moesin) family has attracted our attention.

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The proteins of ERM (Ezrin, Radixin, Moesin) family are general cross-linkers

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between plasma membrane and actin cytoskeleton. These highly homologous proteins are composed by a globular N-terminal FERM (Four-point-one, Ezrin, Radixin,

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Moesin) domain capable to interact directly or indirectly with the transmembrane

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proteins located at plasma membrane (such as CD44 and the ICAMs), followed by a

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central α-helical region, and a hydrophilic C-terminal tail responsible for the binding of F-actin or FERM domain. Represented by the best characterized Ezrin, ERM

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proteins play very important roles in the morphology, transduction of extracellular signals, adhesion and motility of the cells, mainly by regulating the actin filament remodeling which is the key event in most of these processes [18-20]. Notably, Ezrin is a major and critical component in actin-rich structures such as microvilli, focal adhesion, filopodia, lamellipodia and membrane ruffles [21, 22]. Based on the critical role of 14-3-3ζ both in cell migration and in cancer pathogenesis and development, we hypothesized that the presumed interaction between 14-3-3ζ and ezrin may be involved in the regulation of cell migration, and especially in cancer metastasis. 4

ACCEPTED MANUSCRIPT Results 14-3-3ζ interacts with ezrin in cells and in vitro

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We firstly carried out a co-immunoprecipitation experiment using an anti-ezrin

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antibody to confirm the interaction of this protein with 14-3-3ζ in U266 cells. As shown in Fig.1a, endogenous 14-3-3ζ is specifically co-immunoprecipitaed with endogenous ezrin by ezrin antibody. This interaction was then re-confirmed by

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another co-immunoprecipitation assay in HEK293 cells transiently expressing

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HA-14-3-3ζ and FLAG-ezrin tagged proteins. HEK293 cells co-transfected either by FLAG empty vector plus HA-14-3-3ζ recombinant plasmid, or by FLAG-ezrin and

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HA-14-3-3ζ recombinant plasmid were lysed 24 hours after transfection. Cell lysates

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were used for a co-immunoprecipitation assay with an anti-FLAG antibody as

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described in the Materials and Methods. Our results showed that in the cells transfected with FLAG-ezrin and HA-14-3-3ζ expressing vectors, HA-14-3-3ζ was

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co-immunoprecipitated with FLAG-ezrin by FLAG antibody, whereas in those transfected by FLAG empty vector and HA-14-3-3ζ expressing vector, no HA-14-3-3ζ could be detected in the immuno-complex (Fig. 1b). In order to demonstrate the direct interaction between 14-3-3ζ and ezrin by excluding the possibility that other proteins in cell lysates might bridge the binding of these two proteins, we performed an in vitro GST pull down experiment using GST-14-3-3ζ and His tagged-ezrin recombinant proteins. Fig. 1c shows the direct and specific binding of GST-14-3-3ζ and His tagged-ezrin in an acellular context, since His tagged-ezrin could be pulled down by the immobilized GST-14-3-3ζ but not the immobilized GST. 5

ACCEPTED MANUSCRIPT Finally, confocal analysis was used to assess the sub-cellular distribution of 14-3-3ζ and ezrin. As shown in Fig. 2a, a co-localization of 14-3-3ζ and ezrin could be

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observed in tagged forms overexpressed in transfected HEK293 cells. In order to

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demonstrate the sub-cellular distribution of overexpressed 14-3-3ζ and ezrin more clearly, we magnified the images within Fig. 2a, and showed them in Fig. 2b. These

membrane ruffles of HEK293 cells.

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magnified images clearly demonstrated the co-localization of both proteins in the

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Mapping of the binding domains responsible for 14-3-3-ezrin interaction To determine the region of ezrin involved in the interaction with 14-3-3ζ, we

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have constructed five N-terminal FLAG-tagged ezrin truncated fragments, which

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corresponded respectively to the N-terminal plus the central α-helical domains

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(FLAG-Ez-(N+H)), the N-terminal domain (FLAG-Ez-N), the central α-helical domain (FLAG-Ez-H), the C-terminal domain (FLAG-Ez-C), and the central α-helical

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domain followed by the C-terminal domain (FLAG-Ez-(H+C))(Fig. 3a). These ezrin truncated fragments, as well as the FLAG-tagged full-length ezrin (FLAG-EzFL), were transiently expressed in HEK293 cells, followed by GST pull-down assays using the recombinant GST-14-3-3ζ protein to test their binding ability for 14-3-3ζ. As shown in the figure 3b, among these truncated peptides, only the FLAG-Ez-(N+H) construct still retained the ability to interact with 14-3-3ζ. However, neither Ez-N nor Ez-H constructs could bind to 14-3-3ζ, which suggested that the interaction between 14-3-3ζ and ezrin might require an adequate structural conformation provided by the co-existence of the N-terminal and central α-helical domains. Moreover, we used 6

ACCEPTED MANUSCRIPT Co-IP experiment to further confirm that no interaction between Ez-C and 14-3-3ζ was not caused by the underloading of the c-terminus construct. The result showed

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that no interaction between FLAG-Ez-C and HA-14-3-3ζ could be detected whereas it

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was not the case for FLAG-Ez-(N+H) with comparable expression level (Suppl. Fig 1).

Next, we used gel overlay assays to identify the region in 14-3-3ζ which is

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important for the binding to ezrin. The structure of each 14-3-3ζ monomer is

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composed by nine antiparallel α-helices (αA to αI) organized into groups of two, two, two, and three helices [23-25]. The first four helices are involved in dimer formation

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[26], Based on this structural organization, we have constructed and expressed three

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GST fused 14-3-3ζ truncated peptides, respectively corresponding to the αA to αD

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helices (including the amino acids 1-107, indicated hereafter as AD construct), αE to αF helices (EF construct, including the amino acids 108-162), and αG to αI helices

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(GI construct, including the amino acids 163-245) (Figure 3c). E coli cell lysates expressing these GST-14-3-3ζ fragments were subjected to SDS-PAGE, and the resulting protein membranes were incubated with lysates of HEK293 cell expressing either FLAG-EzFL or FLAG-Ez-(N+H) recombinant proteins, as described in Materials and Methods. As shown in Fig.3d, both FLAG-EzFL and FLAG-Ez-(N+H) recombinant proteins bound specifically to GI construct. The ezrin binding region may therefore be located in the αG to αI helices in 14-3-3ζ. 14-3-3ζ and ezrin are both important for efficient cell migration and the formation of membrane ruffle 7

ACCEPTED MANUSCRIPT In the light of previous studies demonstrating independently the involvement of 14-3-3ζ and ezrin in cell migration and tumor metastasis, and the function of ezrin as

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a cross-linker between plasma membrane and actin cytoskeleton, we hypothesized

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that the physical interaction between these two proteins might be important for the function of these two proteins in such cellular and pathological processes. In order to verify this hypothesis, we have either overexpressed HA-14-3-3ζ or knocked down

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ezrin by siRNA in HEK293 cells, and evaluate the migrating ability of the cells by

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using transwell assays (Fig.4 a-c). Our results showed that the overexpression of HA-14-3-3ζ in the cells stimulated significantly the migrating ability whereas

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knocking down ezrin affected greatly this capacity (Fig. 4b-c), indicating that both

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proteins are important for this cellular process. The formation of membrane ruffles is

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a characteristic feature of migrating cells. It has been well documented that ezrin is intimately involved in membrane ruffling [27]. Ezrin is located at membrane ruffles

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and is required for their formation [28-31]. Therefore, we tried to determine if both 14-3-3ζ and ezrin are involved in the formation of membrane ruffles. HEK293 cells were transfected separately or jointly by HA-14-3-3ζ and FLAG-ezrin and then examined by immunofluorescence and con-focal analyses for the formation of membrane ruffles. Fig 4d showed that membrane ruffles (indicated by the arrows) both stained by F-actin and the overexpressed HA-14-3-3ζ or FLAG-ezrin could be observed in cells transfected separately or jointly by HA-14-3-3ζ and FLAG-ezrin, whereas in cells transfected by empty vectors, there was no obvious membrane ruffles. Taken together, these results suggested that 14-3-3ζ and ezrin are both important for 8

ACCEPTED MANUSCRIPT efficient cell migration and the formation of membrane ruffle. The counting of the cells forming membrane ruffles revealed that in the cells transfected by empty vectors,

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only about 5% of them have formed membrane ruffles, whereas in the cells

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transfected either by HA-14-3-3ζ or by FLAG-ezrin, about 20% of them were counted with membrane ruffles, and more than 40% in the cells co-expressing jointly these two proteins (Fig.4e).

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The functions of 14-3-3ζ and ezrin in cell migration and membrane ruffling are

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linked

We next investigated if the functions of 14-3-3ζ and ezrin in the cellular

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migration and the formation of membrane ruffles are linked and mediated by their

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physical interaction, or independent from each other. First, we transiently

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overexpressed HA-14-3-3ζ in HEK293 cells with or without knocking down ezrin expression with siRNA (Fig. 5a), and examined the mobility of these

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siRNA-transfected cells using transwell assays. As expected, ezrin knocking down in HEK293 cells affected considerably their migrating ability, and conversely, 14-3-3ζ overexpression stimulated this ability when ezrin expression was not altered (Fig. 5b and 5c). Interestingly, when ezrin expression was knocked down in the cells overexpressing 14-3-3ζ, the cell migrating ability dropped down dramatically to a level comparable to that of cells treated only with ezrin siRNA. In the other word, knocking down of ezrin expression cancelled almost completely the stimulatory effect of 14-3-3ζ overexpression. Then, we transiently overexpressed FLAG-ezrin in HEK293 cells stably knocked down endogenous 14-3-3ζ expression and examined the 9

ACCEPTED MANUSCRIPT mobilityof the cells using transwell assays. The result showed that the stimulatory effect of ezrin overexpression could be inhibited by knocking down of 14-3-3ζ

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expression (Fig. 5d and 5e). These phenomena might not be the simple compensation

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between the stimulatory effect of overexpression of one protein and the inhibitory effect of the knocking down of the other protein, which would have been independent from each other. In contrast, it supported our hypothesis in which these two regulatory

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effects were linked in the regulation of cell mobility.

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Next, we examined if the stimulation of cell migration by 14-3-3ζ-ezrin interaction was correlated with the formation of membrane ruffles. To this end,

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HEK293 cells treated either with control siRNA or ezrin siRNA, and transiently

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overexpressing HA-14-3-3ζ or not were stained with anti-HA M2 antibody, ezrin

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antibody and rhodamin-labeled phalloidin, then examined by immunofluorescence and con-focal analysis for ezrin and F-actin distribution and cell morphology. As

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shown in Fig. 6a and 6b, 14-3-3ζ overexpression promoted the formation of membrane ruffles (indicated by the arrows), while knocking down of ezrin expression by siRNA abrogated this effect. Consistantly, this result was further confirmed by the result of the experiments in which, conversely, the HEK293 cells with stable knockdown of 14-3-3ζ were transfected with FLAG-ezrin. These results showed that the knockdown of 14-3-3ζ expression inhibited the stimulatory effect of ezrin overexpression (Fig. 6c and 6d). These results suggested that the interaction between 14-3-3ζ and ezrin might be associated with the formation of membrane ruffles during cell migration. In addition, the results presented in Fig. 6a and 6c show that if we 10

ACCEPTED MANUSCRIPT knock down ezrin or 14-3-3ζ by specific siRNA, no more signal can be detected, which demonstrate clearly that the anti-ezrin antibody and the anti-14-3-3ζ antibody

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we used are specific in IF experiments.

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The integrity of ezrin is required to mediate the stimulation of cell migration and membrane ruffling by 14-3-3ζ

Since our GST pull down and gel overlay experiments demonstrated that the

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N+H fragment of ezrin was sufficient for 14-3-3ζ binding, we investigated if it was

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also sufficient to mediate the stimulation of cell migration and membrane ruffling by 14-3-3ζ. HEK293 cells were co-transfected by various combinations of plasmids to

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express, either alone or together with HA-14-3-3ζ, the FLAG-tagged full length ezrin

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protein, the N+H, or C truncated fragments (Fig. 7a). These cells were then analyzed

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for their migrating ability and the formation of membrane ruffles by transwell and confocal analyses as described above. Fig. 7b and 7c show that, when expressed alone,

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neither N+H nor C truncated fragment could promote cell migration as the full-length ezrin did. More interestingly, when co-expressed with 14-3-3ζ, not only these two truncated peptides were unable to enhance the stimulatory effect of 14-3-3ζ as has been observed for the full-length ezrin, they could in contrast inhibit this effect. In particular, the co-expression of N+H fragment with 14-3-3ζ cancelled totally the stimulatory effect of 14-3-3ζ probably by interfering the binding of the overexpressed 14-3-3ζ with endogenous ezrin (Fig. 7c). These results indicated that the integrity of ezrin protein, composed by both N+H and C domains was required to mediate the stimulatory effect of 14-3-3ζ, even though N+H domain alone was sufficient for the 11

ACCEPTED MANUSCRIPT binding with 14-3-3ζ. Consistently, our results of con-focal analysis showed that the integrity of ezrin was also required for the formation of membrane ruffles (Fig. 7d).

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When FLAG-ezrin was co-expressed with HA-14-3-3ζ in HEK293 cells, an enhanced

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membrane ruffling could be observed, compared with those expressing only HA-14-3-3ζ or FLAG-ezrin alone. In contrast, the co-expression of ezrin N+H fragment or C-ter fragment with 14-3-3ζ inhibited the formation of membrane ruffles

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and led to the abnormal actin filaments organization (Fig. 7d and 7e).

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The phosphorylation of Thr567 in ezrin is important for both 14-3-3ζ-ezrin interaction and membrane ruffling

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The threonine 567 of ezrin plays an important role in the function of this protein.

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The phosphorylation of this residue by Thr/Ser kinases such as Rho-kinase, PKC- or

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PKC- can lead to the unfolding, and consequently the activation of ezrin protein under inactive conformation due to its head-to-tail association [32-34]. The relieved

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C-terminal domain can thereby be capable to bind F-actin. In order to investigate if the phosphorylation on ezrin T567 could be important for the interaction between 14-3-3ζ and ezrin and for their involvement in the regulation of cell migration, we have constructed two ezrin point mutants, the ezrin T567A which is the phosphorylation-defective form, and the ezrin T567D mutant in which the threonine 567 residue was replaced by an aspartate micmicking the phosphorylation at this position. Both mutants have been largely used in previous studies by others, and are considered respectively as the inactive and constitutively active forms of ezrin. FLAG-tagged wild type ezrin, T567A or T567D ezrin (referred hereafter to as 12

ACCEPTED MANUSCRIPT FLAG-Ezwt, FLAG-EzT567A, and FLAG-Ez567D) were co-transfected with HA-14-3-3ζ into HEK293 cells. Co-immunoprecipitation experiments were then

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performed to evaluate the binding capacities of these three forms of ezrin with

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14-3-3ζ. Our results demonstrated that FLAG-EzT567D bound to HA-14-3-3ζ much more strongly than FLAG-Ezwt and FLAG-EzT567A (Fig. 8a), suggesting that 14-3-3ζ bound preferentially to phosphorylated ezrin in active conformation.

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The effect of these two mutations on the formation of membrane ruffles was also

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examined by immunofluorescence and con-focal analysis (Fig. 8b). As expected, membrane ruffling was readily observed in HEK293 cells expressing FLAG tagged

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wild type and T567D mutant ezrin (Fig. 8b, indicated by the arrows). The ability of

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FLAG-Ez567D to promote the formation of membrane ruffles was superior to that of

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FLAG-Ezwt, and that FLAG-EzT567A expressing cells were more unlikely to form

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the membrane ruffles (Fig. 8c).

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ACCEPTED MANUSCRIPT Discussion 14-3-3ζ protein, as well as other 14-3-3 subtypes, has been shown to be

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important for a myriad of cellular processes, and to be involved in the genesis and

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development of cancer [3-5]. Particularly, researchers have reported that 14-3-3ζ protein promoted cell migration and cancer metastasis through actin filaments remodeling [35-37]. Although proteomic and confocal microscopy studies have

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provided evidences of 14-3-3 interaction and co-localization with actin filaments

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[38-40], attempts to detect a direct interaction between these two proteins by means of co-sedimentation have revealed to be vain [41]. It is therefore believed that 14-3-3

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proteins may interact with actin filament associated proteins rather than directly with

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F-actin itself, and thereby regulates cell motility. In this study, we provide evidences

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of a direct interaction between 14-3-3ζ and ezrin, an actin filaments binding protein intimately involved in cellular morphology, adhesion, and motility. The

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co-localization of these two proteins has been observed in the cells, particularly, with the bundles of actin filaments in the membrane ruffles of HEK293 cells. One of the possibility is that ezrin might facilitate the interaction between 14-3-3ζ and F-actin at the right place of the cell where the reorganization of actin filaments drives the morphological changes and the movement of the cell. In support of this idea are our results showing that the N-terminal and central helical domains of ezrin (Ez-(N+H)) are responsible for its binding with 14-3-3ζ while its C-terminal region (Ez-C) mediates its binding with F-actin. Furthermore, the integrity of ezrin protein is indispensable for mediating the effect of 14-3-3ζ on cell migration, whereas the 14

ACCEPTED MANUSCRIPT co-expression of either Ez-(N+H) or Ez-C fragment with 14-3-3ζ inhibits the effect of the latter (Fig. 7). Another observation in echo with this argument is the enhanced

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binding of ezrin T567D mutant to 14-3-3ζ compared with wild type ezrin and T567A

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mutant. This suggests that 14-3-3ζ binds preferentially to ezrin protein under “opened” and activated conformation which has also been shown to interact with F-actin. In order to verify the possible targeting of 14-3-3ζ to the membrane ruffles by ezrin, we

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have overexpressed either the wild type ezrin or the N+H domain of ezrin in HEK293

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cells, and then analyzed by cell fractionation and con-focal experiments the distribution of 14-3-3 in the membrane. As shown in Supplementary Fig 2a, our

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results by cell fractionation showed that no obvious variation could be observed for

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14-3-3 level in the membrane fractions whether if ezrin or ezrin N+H domain was

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overexpressed or not. However, in con-focal experiments, the overexpression of Flag-ezrin in the cells could induce both the formation of membrane ruffles and the

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localization of the endogenous 14-3-3 in this structure (Suppl. Fig 2b, middle panels, indicated by arrows). In contrast, the result of Flag-Ez (N+H) overexpression is hard to interpret in that it could stimulate the formation of some spike form protrusive structures resembling filopodia but without the localization of endogenous 14-3-3 in such structures (Suppl. Fig 2b, lower panels, indicated by arrows). Same result has been observed for the overexpressed 14-3-3 as showed in Fig. 7d. Taken together, our results do not allow the clear confirmation of the direct role of the interaction between 14-3-3 and ezrin for the membrane localization of 14-3-3. 14-3-3 proteins have also been shown to bind other F-actin associated proteins such as Cofilin, the 15

ACCEPTED MANUSCRIPT largest subunit of the myosin light chain phosphatase (MLCP) and the testicular protein kinase (TESK) [2]. The complexity and dynamicity of such a large complex of

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proteins might make it difficult to further demonstrate more clearly the role of ezrin in

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the indirect interaction of 14-3-3 and F-actin filament using the classical biochemical approaches.

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On the other hand, by interaction with ezrin, 14-3-3 could possibly play the role of an adapter protein. For example，the dimer of 14-3-3might connect ezrin to other

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proteins involved in the regulation of actin filament remodeling. In this regard, the

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functional interaction of ezrin and the Rho GTPases (Rho/Rac/cdc42) could be

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considered. The Rho GTPases are intimately involved in the actin filament remodeling and cellular polarization and migration [42-45], and ezrin and other ERM

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proteins are able to regulate the activity of Rho GTPases which has also been shown to be controlled by the phosphorylation by the kinases such as PKA [19, 22, 46-49].

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Moreover, ezrin has been reported to be a potential AKAP (A-Kinase Anchoring Protein) with a PKA RII-binding motif identified within its central α-helical region [50, 51]. The mechanism of activation of Rho GTPases by ERM proteins remains to be unclear, and no direct physical interaction between ezrin and the Rho/Rac/cdc42 proteins has yet been reported. All these arguments converge towards a model in which the interaction between ezrin and 14-3-3 could connect the PKA and Rho GTPases proteins, and thereby regulate their activity. Nevertheless, in this case, it is primordial to be able to detect the direct or indirect interaction between 14-3-3 and

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ACCEPTED MANUSCRIPT the Rho/Rac/cdc42 proteins, and also the existence of a complex comprising all these

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proteins. This requires further investigations.

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ACCEPTED MANUSCRIPT Materials and methods Antibodies and reagents

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Control mouse IgG, 14-3-3ζ mAb and ezrin mAb were obtained from Santa Cruz

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Biotechnology (Santa Cruz, CA); Anti-FLAG M2 mAb and anti-HA 12CA5 mAb were purchased from Sigma-Aldrich (St. Louis, MO); Anti-β-actin mAb was from Proteintech Group, Inc. (Chicago, USA); Anti-His mAb was from Tiangen Biotech

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(Beijing, China). HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat

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anti-mouse IgG were from Jackson ImmunoResearch (West Grove, PA); Alexa Fluor 488 goat anti-mouse IgG, Alexa Flour 596 goat anti-rabbit IgG and Alexa Fluor 405

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(Nantong, Jiangsu, China).

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goat anti-rabbit IgG were obtained from Beyotime Institute of Biotechnology

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Cell culture

HEK293 cells were cultured in DMEM, and human multiple myeloma U266

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cells were cultured in RPMI-1640 medium. All media were supplemented with 10% (v/v) FBS and streptomycin-kanamycin. All cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Plasmid construction pcDNA3-FLAG-ezrin was kindly provided by Philip W. Hinds (Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts). pCS2+-14-3-3ζ was kindly provided by Feng-Qian Li (Department of Pharmocological Sciences, State University of New York, NY). pGEX-14-3-3ζ and pGEX-6P-1 were kindly provided by Helen Piwnica-Worms (Washington University 18

ACCEPTED MANUSCRIPT School of Medicine, St. Louis). To generate a C-terminal His-tagged ezrin expression plasmids the PCR product of ezrin cDNA was cloned into the EcoRV and XhoI site of

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pET-20b(+). Point mutants and deletion mutants of ezrin were generated by PCR, and

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cloned into the EcoRV and XhoI sites of pCMV-FLAG, to produce various ezrin mutants tagged with a FLAG tag at the N terminus. Deletion mutants of 14-3-3ζ were generated by PCR and cloned into EcoRI and SalI sites of pGEX-6P-1, resulting in

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the N terminal GST-tagged 14-3-3ζ constructs. All recombinant plasmids were

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verified by DNA sequencing.

Expression and purification of recombinant proteins

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pET-20b (+) constructs encoding C-terminal His-tageged ezrin were transformed

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into E. coli. Rosetta bacteria. Transformed bacteria were grown to an OD600 of

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0.6-0.8, and the expression of recombinant protein was induced by the addition of IPTG (1µM). After overnight incubation at room temperature, cells were harvested by

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centrifugation at 10000 rpm, 10 min and rinsed twice with PBS. The pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH7.6, 150 mM NaCl, 1% Triton X-100, 5 mM imidazole, 1 mM PMSF, 1×proteinase inhibitor cocktail, lysozyme) for 10 minutes, sonicated to lyse the bacteria, and centriguged (12000 rpm, 4°C for 10 min). The supernatant was applied to a Ni-NTA-agarose column equilibrated in lysis buffer. After elution

with lysis buffer containing 10-50 mM imidazole, bound ezrin

proteins were collected in 1.5 mL EP tube and store at 4°C. GST-14-3-3ζ wild type or deletion mutants were produced in E. coli DH5 strain as described above for the His-tagged ezrin recombinant proteins. After induction with IPTG, the pellets of 19

ACCEPTED MANUSCRIPT cells expressing the appropriate recombinant peptides were resuspended in lysis buffer (50 Mm Tris-HCl, pH7.6, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF,

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1×proteinase inhibitor cocktail, 50 μg/mL lysozyme) for 10 min. After ultrasonication

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and centrifugation at 12000 rpm, 4°C for 10 min, purification of GST-tagged proteins was performed with GST-Sepharose (GE Healthcare). Cell transfection

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For small interference RNA experiments, U266 and HEK293 cells were seeded

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sparsely 24 hours before transfection, and then transfected with ezrin siRNA (100 nM) or no target siRNA according to the manufacturer’s protocol. For overexpression

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experiments, HEK293 cells were transfected with appropriate plasmids at the final

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concentration of 1.5 μg/mL using Lipofectamine 2000 or LTX respectively. For

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experiments where cells were subjected both to siRNA and overexpression treatments, cells were transfected with siRNA 24 h before the transfection of the appropriate

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plasmids as described here above. Western blotting

Proteins were separated using 10% or 12% SDS-PAGE gels followed by eletro-blotting to PVDF membrane using a transfer buffer (25 mM Tris, 192 mM glycine, 0.01% SDS, and 20% methol). Membranes were blocked with 5% (v/v) non-fat milk for 1 h at room temperature. Blots were incubated with the primary antibody overnight at 4°C. After washing, blots were probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies, and then signals were visualized using the ECL detection system, excepted for the detection of 20

ACCEPTED MANUSCRIPT HA-14-3-3ζ and full-length or truncated FLAG-ezrin constructs in transfected HEK293 cells in the experiment presented in Fig. 4a, where DAB staining was used

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for a better visibility.

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Immunofluorescence

Cells grown on glass coverlips were fixed with 4% (v/v) paraformaldehyde or methanol for 30 min at room temperature. After treated with 0.1% (v/v) TritonX-100

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for 10 min at room temperature, they were blocked with 10% (v/v) goat normal serum

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for 1 h and incubated with the primary antibodies overnight at 4°C. After washing with PBS containing 0.05% Tween-20 and 1% BSA, the cells were incubated with the

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indicated secondary antibodies. F-actin was stained using rhodamine-conjugated

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phalloidin. Images of cells mounted at room temperature in 90% glycerol in PBS were

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acquired using confocal microscope with a Plan Neofluar 40×NA 0.75 or 63×NA 1.25 objective. Images were prepared with ImageJ software.

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100 cells expressing the transfected genes were examined and cells with membrane ruffles were scored under an inverted fluorescence microscope using the 20× objective.

Co-immunoprecipitation For transfected HEK293 cells, cells were transfected with the plasmids 48 h before the co-immunoprecipitation experiments. Cells were rinsed with PBS twice and lysed in buffer (20 mM Tris, pH7.5, 150 mM NaCl, 1% Triton X-100, 1× proteinase inhibitor cocktail, 1 mM sodium fluoride, and 1 mM sodium orthovanadate) for 30 min at 4 °C. The lysates were clarified by centrifugation at 13200 rpm for 20 21

ACCEPTED MANUSCRIPT min at 4°C. Protein concentrations were determined using the BCA protein assay kit. After pre-clearing, total protein (1 mg) was incubated with the appropriate primary

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antibody overnight, and Protein A/G PLUS-Agarose beads (20 μL) for 4 h at 4 °C.

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The immunoprecipitates and lysate were subjected to western blot using the antibody indicated. Blot overlay

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After induction with IPTG, cell extracts of E. coli expressing the indicated

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recombinant proteins were separated by SDS-PAE and transferred on to PVDF membranes. Blots were incubated overnight at 4 ° C with the lysate of cells

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expressing FLAG-Ez FL or FLAG-Ez (N+H), and subsequently with anti-FLAG

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antibody at 4°C overnights. After washing twice, the membranes were incubated

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with anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase, and proteins were detected by ECL.

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Cell fractionation

1×107 cells were re-suspended in 1 mL extraction buffer (10 mM Tris-HCl, pH 7.6, 10 mM KCl, 5 mM MgCl2) with protease inhibitors (1 mM PMSF, 1×proteinase inhibitor cocktail), incubated on ice for 10 min, and then lysised by addition of Triton X-100 to the final concentration of about 0.3% (w/v). To check the efficiency of homogenization, 2-3 μL of the homogenized suspension were pipetted onto a cover slide, stained with methylene blue solution and observed under a microscope. If about 80% of the nuclei are not surrounded by cytoplasm, 0.5 volume of nuclei isolation buffer (10 mM Tris-HCl, pH 7.6, 10 mM KCl, 5 mM MgCl2, 0.35M sucrose) was 22

ACCEPTED MANUSCRIPT slowly added to the bottom of extraction buffer, and then nuclei were isolated by centrifugation at 600 g for 10 min. heavy membrane fraction containing mitochondria

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was further obtained by centrifugation at 10000 g for 30 min. 4 volumes of acetone

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were added to cytoplasmic fraction to precipitate cytoplasmatic proteins overnight at -20 ℃. Cytoplasmatic proteins were obtained by centrifugation at 10000 g for 30 min. The cellular components were added with SDS buffer for western blot.

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Inhibition of 14-3-3ζ by RNA interference

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To stably knock down endogenous 14-3-3ζ expression, we used lentivirus packing shRNA expression vector (GenePharma, Shanghai, China) to infect cells.

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Target cells were infected with lentivirus for 24-48 h according to manufacturer’s

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instruction. The RNAi oligonucleotide sequence used to knock down endogenous

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14-3-3ζ expression is as follows: ACGGTTCACATTCCATTAT. The sequence of TTCTCCGAACGTGTCACGTTTC was used as negative control.

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Cell migration assay

Transfection was performed using Lipofectamine 2000 2 days before assay. Cells (2.5×105) were seeded in the upper chamber and incubated for 36 h. The cells were then fixed with methanol and stained with crystal violet. For the quantitation of migrated cells, 5 random fields of migrated cells in each well were counted under a microscope. Statistical analysis Statistical analysis was carried using Student’s t-test. Data were expressed as mean ± SEM of 3 independent experiments, with p-value less than 0.05 considered 23

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significant.

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ACCEPTED MANUSCRIPT Acknowledgements We thank Dr. Philip W. Hinds (Molecular Oncology Research Institute, Tufts

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Medical Center, USA) for providing pcDNA3-FLAG-ezrin, Dr. Feng-Qian Li

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(Department of Pharmocological Sciences, State University of New York, NY, USA) for providing pCS2+-14-3-3ζ and Dr. Helen Piwnica-Worms (Washington University School of Medicine, USA) for providing pGEX-14-3-3ζ and pGEX-6P-1. The

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following grants have supported this work: National Program on Key Basic Research

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Project (973 Program) (Grant No.2011CB910700), High-Level Talents Project of the Universities of Guangdong (No. [2011]431), National Natural Science Foundation of

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China (Grant No. 31000628), Fundamental Research Funds for the Central

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Universities (Grant No. 21611430; 21610101; 21609317), and Natural Science

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.

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Foundation of Guangdong Province (Grant No. S2013030013315).

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ACCEPTED MANUSCRIPT References [1] Zhao J, Meyerkord CL, Du Y, Khuri FR, Fu H. 14-3-3 proteins as potential therapeutic targets. in Seminars in cell & developmental biology Elsevier 2011.

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[2] Sluchanko N, Gusev N. (2010) 14-3-3 proteins and regulation of cytoskeleton. Biochemistry (Moscow) 2010; 75:1528-46.

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[3] Neal CL, Yu D. 14-3-3ζ as a prognostic marker and therapeutic target for cancer. Expert opinion on therapeutic targets 2010;14:1343-54. opinion on therapeutic targets 2012;16:515-23.

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[4] Matta A, Siu KM, Ralhan R. 14-3-3 zeta as novel molecular target for cancer therapy. Expert [5] Yang X, Cao W, Zhang L, Zhang W, Zhang X, Lin H. Targeting 14-3-3zeta in cancer therapy. Cancer gene therapy 2012;19:153-9.

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[6] Ge F, Lu XP, Zeng HL, He QY, Xiong S, Jin L, et al. Proteomic and functional analyses reveal a dual molecular mechanism underlying arsenic-induced apoptosis in human multiple myeloma cells. Journal of proteome research 2009;8:3006-19.

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[8] Somiari RI, Somiari S, Russell S, Shriver CD. Proteomics of breast carcinoma. Journal of

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[9] Shoji M, Kawamoto S, Setoguchi Y, Mochizuki K, Honjoh T, Kato M, et al. The 14-3-3 protein as the antigen for lung cancer-associated human monoclonal antibody AE6F4. Human antibodies and hybridomas 1993;5:123-30.

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[10] Qi W, Liu X, Qiao D, Martinez JD. Isoform-specific expression of 14-3-3 proteins in human lung cancer tissues. International journal of cancer 2005;113:359-63. [11] Bajpai U, Sharma R, Kausar T, Dattagupta S, Chattopadhayay T, Ralhan R. Clinical significance of 14-3-3 zeta in human esophageal cancer. The International journal of biological markers

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[12] Arora S, Matta A, Shukla NK, Deo S, Ralhan R. Identification of differentially expressed genes in oral squamous cell carcinoma. Molecular carcinogenesis 2005;42:97-108. [13] Jang JSJ, Cho HY, Lee YJ, Ha WS, Kim HW. The differential proteome profile of stomach cancer: identification of the biomarker candidates. Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics 2004;14:491-9. [14] Chatterjee D, Bai Y, Wang Z, Beach S, Mott S, Roy R, et al. RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis. Journal of Biological Chemistry 2004;279:17515-23. [15] Huber E, Vlasny D, Jeckel S, Stubenrauch F, Iftner T. Gene profiling of cottontail rabbit papillomavirus-induced carcinomas identifies upregulated genes directly Involved in stroma invasion as shown by small interfering RNA-mediated gene silencing. Journal of virology 2004;78:7478-89. [16] Neal CL, Yao J, Yang W, Zhou X, Nguyen NT, Lu J, et al. 14-3-3ζ overexpression defines high risk for breast cancer recurrence and promotes cancer cell survival. Cancer research 2009;69:3425-32. [17] Fan T, Li R, Todd NW, Qiu Q, Fang HB, Wang H, et al. Up-regulation of 14-3-3ζ in lung cancer and its implication as prognostic and therapeutic target. Cancer research 2007;67: 7901-6. 26

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[20] Neisch AL, Fehon RG. Ezrin, radixin and moesin: key regulators of membrane–cortex interactions and signaling. Current opinion in cell biology2011; 23:377-82.

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[22] Pujuguet P, Del Maestro L, Gautreau A, Louvard D, Arpin M. Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation. Molecular biology of the cell 2003;14:2181-91.

[23] Danielle MW, Heath E, Katy LG, Huanqin D, Haian F, Joanna MW, et al. NMR spectroscopy of

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14-3-3zeta reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3zeta. Biochemical Journal 2011;437: 493-503. [24] Xiao B, Smerdon SJ, Jones DH, Dodson GG, Soneji Y, Aitken A, et al. Structure of a 14-3-3 and

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[26] Yang X, Lee WH, Sobott F, Papagrigoriou E, Robinson CV, Grossmann JG, et al. Structural basis

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for protein–protein interactions in the 14-3-3 protein family. Proceedings of the National Academy of Sciences 2006;103:17237-42. [27] Borm B, Requardt RP, Herzog V, Kirfel G. Membrane ruffles in cell migration: indicators of

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inefficient lamellipodia adhesion and compartments of actin filament reorganization. Exp Cell Res 2005; 302:83-95.

[28] Franck Z, Gary R, Bretscher A. Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable. Journal of cell science 1993;105:

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[29] Amieva MR, Furthmayr H. Subcellular localization of moesin in dynamic filopodia, retraction fibers, and other structures involved in substrate exploration, attachment, and cell-cell contacts. Experimental cell research 1995;219:180-96. [30] Bretscher A, Reczek D, Berryman M. Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. Journal of cell science 1997;110:3011-8. [31] Menager C, Vassy J, Doliger C, Legrand Y, Karniguian A. Subcellular localization of RhoA and ezrin at membrane ruffles of human endothelial cells: differential role of collagen and fibronectin. Experimental cell research 1999;249:221-30. [32] Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K, et al. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. The Journal of cell biology 1998;140:647-57. [33] Oshiro N, Fukata Y, Kaibuchi K. Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. Journal of Biological Chemistry 1998;273:34663-6. [34] Wald F, Oriolo A, Mashukova A, Hamid A, Salas P. Ezrin phosphorylation in T567 is mediated by 27

ACCEPTED MANUSCRIPT atypical PKC in intestinal epithelial cells: P‐101. Inflammatory Bowel Diseases 2008;14:S38. [35] Goc A, Abdalla M, Al-Azayzih A, Somanath PR. Rac1 activation driven by 14-3-3ζ dimerization promotes prostate cancer cell-matrix interactions, motility and transendothelial migration. PloS one 2012;7: e40594.

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[36] Bergamaschi A, Frasor J, Borgen K, Stanculescu A, Johnson P, Rowland K, et al. 14-3-3ζ as a predictor of early time to recurrence and distant metastasis in hormone receptor-positive

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and-negative breast cancers. Breast cancer research and treatment 2013;137:689-96. [37] Lu J, Guo H, Treekitkarnmongkol W, Li P, Zhang J, Shi B, et al. 14-3-3ζ cooperates with ErbB2

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[38] Rubio M, Geraghty K, Wong B, Wood N, Campbell D, Morrice N, et al. 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular

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metabolism, proliferation and trafficking. Biochem. J 2004;379;395-408. [39] Liang S, Yu Y, Yang P, Gu S, Xue Y, Chen X. Analysis of the protein complex associated with 14-3-3 epsilon by a deuterated-leucine labeling quantitative proteomics strategy. Journal of

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[40] Chen XQ, Yu AC. The association of 14-3-3gamma and actin plays a role in cell division and apoptosis in astrocytes. Biochemical and biophysical research communications 2002;296: 657-63. [41] Birkenfeld J, Betz H, Roth D. Identification of cofilin and LIM-domain-containing protein kinase

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[42] Frame MC, Brunton VG. Advances in Rho-dependent actin regulation and oncogenic transformation. Current opinion in genetics & development 2002;12:36-43. [43] Fukata M, Nakagawa M, Kaibuchi K. Roles of Rho-family GTPases in cell polarisation and

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directional migration. Current opinion in cell biology 2003;15:590-7. [44] Mack NA, Whalley HJ, Castillo-Lluva S, Malliri A. The diverse roles of Rac signaling in tumorigenesis. Cell Cycle 2011;10:1571. [45] O'Connor KL, Chen M. Dynamic functions of RhoA in tumor cell migration and invasion. Small

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[46] Howe AK. Regulation of actin-based cell migration by cAMP/PKA. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2004;1692:159-74. [47] Prag S, Parsons M, Keppler MD, Ameer-Beg SM, Barber P, Hunt J, et al. Activated ezrin promotes cell migration through recruitment of the GEF Dbl to lipid rafts and preferential downstream activation of Cdc42. Molecular biology of the cell 2007;18:2935-48. [48] Ivetic A, Ridley AJ. Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes. Immunology 2004;112:165-76. [49] Hughes SC, Fehon RG. Understanding ERM proteins–the awesome power of genetics finally brought to bear. Current opinion in cell biology 2007;19:51-6. [50] Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, Mangeat PH, et al. Ezrin is a cyclic AMP ‐dependent protein kinase anchoring protein. The EMBO Journal 1997;16:35-43. [51] Schillace RV, Miller CL, Carr DW. AKAPs in lipid rafts are required for optimal antigen presentation by dendritic cells. Immunology and cell biology 2011;89:650-8.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1 14-3-3ζ interacts with ezrin in U266 and transfected 293T cells and in vitro

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a) Co-immunoprecipitation experiments have been performed using U266 cell lysates

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with anti-ezrin antibody, or with non-immune IgG as negative control. Immunocomplexes (lanes 1 and 2), together with the whole cell lysates (lanes 3 and 4) were analyzed by western blotting with anti-14-3-3ζ and anti-ezrin antibodies. b)

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HEK293 cells were co-transfected with the plasmids pcDNA3 and pCS2+-14-3-3ζ

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(lane 1 and 3), or with pcDNA3-FLAG-ezrin and pCS2+-14-3-3ζ (lane 2 and 4). 48 h after transfection, immunoprecipitation experiments were performed with cell lysates

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using anti-HA antibody. Immunoprecipitates (lane 1 and 2) and the whole cell lysates

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(lane 3 and 4) were probed respectively with anti-HA and anti-FLAG antibodies. c)

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Purified GST (lane 1) or GST-14-3-3ζ recombinant proteins (lane 2) were immobilized on Sepharose-Glutathione beads and incubated with purified His-tagged

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ezrin protein. Lane 3 represents 1% of input His-tagged ezrin. The amounts of GST and GST-14-3-3ζ used in the assays were checked by coomassie blue staining (lower panel).

Fig. 2 Co-localization of 14-3-3ζ and ezrin in transfected HEK293 cells a) HEK293 cells co-transfected with pcDNA3-FLAG-ezrin and pCS2+-14-3-3ζ were treated with the appropriate anti-FLAG M2 mAb and anti-HA 12CA5 mAb primary antibodies and the corresponding secondary antibodies. HA-14-3-3ζ and FLAG-ezrin were respectively colored in red and green. Arrows indicate the co-localization of 14-3-3ζ and ezrin in the membrane ruffle-like structures. Bar, 10 μm. b) The detail of 29

ACCEPTED MANUSCRIPT the co-localization of HA-14-3-3ζ and FLAG-ezrin in membrane ruffles. Bar, 10 μm. Fig. 3 Mapping of binding domains in 14-3-3ζ and ezrin

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a) Construction of ezrin truncated fragments. Ezrin fragments corresponding to

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various regions of ezrin as illustrated in the diagram have been amplified by PCR and cloned into pcDNA3-FLAG vector then expressed in HEK293 cells. b) GST-pull down assays using GST-14-3-3ζ recombinant protein and the indicated ezrin

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fragments tagged to FLAG were performed as described in Materials and Methods.

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Western blotting with anti-FLAG antibody was used to assess the binding of the ezrin fragments to GST-14-3-3ζ (the upper left panel). The equal amount of GST-14-3-3ζ

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used was checked with anti-GST antibody (the lower left panel). The input amount of

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ezrin truncated fragments were shown in the right panel by Western blotting using the

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anti-FLAG antibody. c) Construction of 14-3-3ζ truncated fragments. 14-3-3ζ fragments corresponding respectively to the helices A-D, helices E-F, or helices G-I of

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14-3-3ζ have been amplified by PCR and cloned into pGEX vector to produce in E. coli the corresponding GST recombinant proteins. d) Protein extracts from E. coli bacteria transfected by various constructions of 14-3-3ζ fragments and induced or not with 1 mM IPTG as described have been separated by SDS-PAGE and transferred onto a PVDF membrane, which was then incubated with the cell lysates of HEK293 cells expressing either full-length FLAG-ezrin (FLAG-EzFL) or FLAG-ezrin (N+H). The binding was then revealed by the anti-FLAG M2 primary antibody coupled to the appropriate secondary antibody. Fig. 4 14-3-3ζ and ezrin are both important for efficient cell migration and 30

ACCEPTED MANUSCRIPT membrane ruffling a) HEK293 cells were transfected with pCS2+-14-3-3ζ or ezrin siRNA. 48 h after

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transfection, 14-3-3ζ and ezrin expression were revealed by Western blotting. β-actin

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from the same loading was used as internal control. b) HEK293 cells transfected with pCS2+-14-3-3ζ or ezrin siRNA for 24 h were subjected to transwell assays. 36 h after seeding, migrated cells in randomly chosen fields of the lower chamber were

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photographed and counted. c) Quantification of the migrated cells in transwell assays.

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The mean values of the numbers of migrated cells in 5 fields randomly chosen are presented. And all values are presented as the ratios of the control group’s value. d)

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Subcellular distribution of the transiently expressed HA-14-3-3ζ, FLAG-ezrin and

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F-actin. HEK293 cells were transfected separately or jointly by HA-14-3-3ζ and

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FLAG-ezrin. 48 h after transfection, cells were fixed with 4% paraformaldehyde, treated with 0.1% Triton-100, and incubated with anti-FLAG M2 mAb or anti-HA

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12CA5 mAb primary antibodies and the corresponding secondary antibodies. HA-14-3-3ζ and FLAG-ezrin were colored in blue and green, respectively. F-actin was stained using rhodamine-conjugated phalloidin and colored in red fluorescence. Arrow indicates the membrane ruffles formed in the cells. Bar, 10 μm. e) Quantification of the cells with membrane ruffles. HA-14-3-3ζ and FLAG-ezrin were transiently expressed and the cells were stained as described in d). 100 cells expressing the indicated proteins were examined and cells with membrane ruffles were scored under an inverted fluorescence microscope. The results shown are the means±SD of three independent experiments. *, P < 0.05 compared with the control. 31

ACCEPTED MANUSCRIPT Fig. 5 The functions of 14-3-3ζ and ezrin in cell migration are linked a) Overexpression of HA-14-3-3ζ and knockdown of ezrin in HEK293 cells. HEK293

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cells were either co-transfected or transfected independently by pCS2+-14-3-3ζ and

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ezrin siRNA. The expression level of indicated proteins was then examined by Western blotting with appropriate antibodies. b) HEK293 cells treated as indicated were subjected to transwell assays. 36 h after seeding, migrated cells in randomly

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chosen fields of the lower chamber were photographed and counted. c) Quantification

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of the migrated cells in transwell assays. The mean values of the numbers of migrated cells in 5 fields randomly chosen are presented. All values are presented as the ratios

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of the control group’s value. *, P < 0.05. d) Overexpression of FLAG-ezrin and

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knockdown of 14-3-3ζ in HEK293 cells. HEK293 cells with 14-3-3ζ stably knocked

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down were transiently transfected with FLAG-vector or FLAG-ezrin and subjected to transwell assays. 36 h after seeding, migrated cells in randomly chosen fields of the

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lower chamber were photographed and counted. e) Quantification of the migrated cells in transwell assays. The mean values of the numbers of migrated cells in 5 fields randomly chosen are presented. All values are presented as the ratios of the control group’s value. *, P < 0.05. Fig. 6 The interaction between 14-3-3ζ and ezrin was associated with the formation of membrane ruffle during cell migration a) Subcellular localization of the transiently expressed HA-14-3-3ζ, and the endogenous ezrin and F-actin. HA-14-3-3ζ and ezrin were respectively colored in blue and green. F-actin was stained using rhodamine-conjugated phalloidin and 32

ACCEPTED MANUSCRIPT colored in red fluorescence. Arrow indicates the membrane ruffles formed in the HA-14-3-3ζ overexpressing cells. Bar, 10 μm. b) Quantification of the cells with

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membrane ruffles. The cells were stained as described in a). 100 cells expressing the

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indicated proteins were examined and cells with membrane ruffles were scored under an inverted fluorescence microscope. The results shown are the means±SD of three independent experiments. *, P < 0.05. c) Subcellular localization of the transiently

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expressed FLAG-ezrin, and the endogenous 14-3-3ζ and F-actin. GFP, 14-3-3ζ and

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FLAG-ezrin were respectively colored in green, blue and red. Arrow indicates the membrane ruffles formed in the FLAG-ezrin overexpressing cells. Bar, 10 μm. d)

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Quantification of the cells with membrane ruffles. The cells were stained as described

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in c). 100 cells expressing the indicated proteins were examined and cells with

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membrane ruffles were scored under an inverted fluorescence microscope. The results shown are the means±SD of three independent experiments. *, P < 0.05.

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Fig. 7 The integrity of ezrin is required for the stimulation of cell migration and membrane ruffling by 14-3-3ζ a) HEK293 cells expressing various combinations of HA-14-3-3ζ and full-length or truncated ezrin were subjected to Western blotting using the indicated antibodies. b) Representative fields of migrated cells expressing various combinations of HA-14-3-3ζ and full-length or truncated ezrin in transwell assays. c) Quantification of the migrated cells in transwell assays, with the statistical analysis carried out using Student’s t-test. The mean values of cell numbers counted in 5 fields are presented. And all values are presented as the ratios of the control group’s value. d) Subcellular 33

ACCEPTED MANUSCRIPT distribution of transiently expressed HA-14-3-3ζ, full-length FLAG-Ez or FLAG-Ez (N+H) fragment, and endogenous F-actin in HEK293 cells. HA-14-3-3ζ was colored

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in blue. FLAG-Ez, FLAG-Ez (N+H) fragment and FLAG-Ez C were colored in green.

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F-actin was stained using rhodamine-conjugated phalloidin and colored in red fluorescence. Arrows denote the membrane ruffles formed in the cells co-expressing HA-14-3-3ζ and FLAG-Ez wt proteins. Bar, 10 μm. e) Quantification of the cells with

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membrane ruffles. The cells were stained as described in d). 100 cells expressing the

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indicated proteins were examined and cells with membrane ruffles were scored under an inverted fluorescence microscope. The results shown are the means±SD of three

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and FLAG-Ez FL.

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Fig. 8 The phosphorylation of Thr567 in ezrin is important for 14-3-3ζ-ezrin interaction, membrane ruffling and cell migration

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a) Co-immunopreciptation experiments in cell lysates of 293T cells transiently co-expressing HA-14-3-3ζ and FLAG-ezrin wt/T567A/T567D. Anti-HA antibody was used for the immunoprecipitation experiments. The binding of the wild type or T567 mutants to HA-14-3-3ζ was checked by Western blotting using the indicated antibodies. b) Subcellular distribution of transiently expressing HA-14-3-3ζ, FLAG-Ez wt or T567A/T567D, and F-actinin HEK293 cells. HA-14-3-3ζ was colored in blue. FLAG-Ez wt or T567A/T567D was colored in green. F-actin was stained using rhodamine-conjugated phalloidin and colored in red. Arrows indicate the membrane ruffles formed in the cells. Bar, 10 μm. c) Quantification of the cells with 34

ACCEPTED MANUSCRIPT membrane ruffles. The cells were stained as described in b). 100 cells expressing the indicated proteins were examined and cells with membrane ruffles were scored under

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an inverted fluorescence microscope. The results shown are the means±SD of three

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independent experiments. *, P < 0.05 compared with the control.

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ACCEPTED MANUSCRIPT Supplementary Figure Legends Supplementary Fig.1 No interaction between FLAG-Ez-C and HA-14-3-3ζ was

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Supplementary Fig.2 The distribution of 14-3-3ζ in membrane and cytosolic

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a) HEK293 cells transfected ezrin siRNA, FLAG-vector, FLAG-Ez FL or FLAG-Ez (N+H) were subjected to cell fractionation and western blotting using the indicated

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antibodies. C, cytosolic extract, N, nuclear extract, M, membrane extract. b) Subcellular distribution of endogenous 14-3-3ζ in HEK293 cells transfected with FLAG-vecotr, FLAG-Ez FL or FLAG-Ez (N+H) fragment. Endogenous 14-3-3ζ was colored in red. FLAG-Ez FL, FLAG-Ez (N+H) were colored in green. Arrows denote the membrane ruffles formed in the cells expressing FLAG-Ez FL or some spike form protrusive structures in the cells expressing FLAG-Ez (N+H). Bar, 10 μm.

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Graphical abstract

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Highlights

14-3-3ζ interacts directly with ezrin in vitro and in the cells.

2.

14-3-3ζ-ezrin interaction promotes membrane ruffling.

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14-3-3ζ-ezrin interaction is important for cell migration.

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Ezrin T567 phosphorylation promotes 14-3-3ζ-ezrin interaction and membrane ruffling.

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