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FEBS Letters 586 (2012) 3090–3096

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Annexin A9 is a interacting partner in membrane-targeted cytoskeletal linker complexes ⇑ Veronika Boczonadi 1, Arto Määttä

School of Biological and Biomedical Sciences, Durham University, DH1 3LE Durham, United Kingdom

article info abstract

Article history: Periplakin regulates organisation and participates in the assembly of epidermal cornified Received 3 April 2012 envelopes. A proteomic approach identified annexin A9 as a novel interacting partner for periplakin Revised 13 July 2012 N-terminus. The presence of annexin A9 in complexes with periplakin was confirmed by immuno- Accepted 16 July 2012 blotting of immunoprecipitated by anti-HA or anti-annexin A9 antibodies. Both endoge- Available online 24 July 2012 nous and GFP-tagged annexin A9 co-localise with endogenous periplakin and transfected Edited by Gianni Cesareni periplakin N-terminus at MCF-7 cell borders and aggregate after Okadaic acid treatment. Annexin A9 and periplakin co-localise in the epidermis and annexin A9 is up-regulated in differentiating keratinocytes, but the epidermal annexin A9 expression does not require periplakin. Keywords: Periplakin Annexin Structured summary of protein interactions: Epidermis Annexin-9 physically interacts with Periplakin by anti bait co-immunoprecipitation (View interaction) Epithelia Periplakin physically interacts with Annexin-9 by anti tag co-immunoprecipitation (View Interaction: 1, 2) Co-immunoprecipitation Periplakin and Annexin-9 colocalize by fluorescence microscopy (View Interaction: 1, 2, 3) Epidermal barrier Keratin-8 and Periplakin colocalize by fluorescence microscopy (View interaction)

Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction an initial scaffold under the plasma membrane upon which a large number of small proteins such as loricrin gets deposited and cross- Periplakin is a member of the plakin family of cytoskeletal linker linked [7,8]. Periplakin is also a major cross-linking platform for the proteins that connect intermediate filaments to and microtu- ceramide lipids that replace membrane phospholipids at the stra- bule cytokeletons and to cell adhesion junctions. Periplakin shows a tum corneum [9]. Even though periplakin targeted mice do restricted tissue distribution, including both stratified and simple not show a barrier defect [10], the ablation of the all three scaffold epithelial tissues and the brain. In the epidermis of the skin, peripla- proteins, periplakin, and involucrin, results in impaired kin expression is upregulated upon keratinocyte differentiation and barrier function and immune cell infiltration to the epidermis [7]. the protein has been shown to associate with desmosomes, cortical The N-terminus of periplakin shares the conserved structure of actin networks and the plasma membrane surrounding desmo- a ‘plakin box’ but lacks the canonical actin-binding domain present somes, while the C-terminus of the protein binds intermediate in the N-terminus of some of the other family members [1]. filaments [1–3]. Notably, autoantibodies against periplakin and Although the crystal structure of periplakin N-terminus has not other plakin family members are found in patients with paraneo- been solved, it is likely to contain an array of individual spectrin- plastic pemphigus [4,5]. related domains that flank a central SH3 domain as seen in two During the epidermal differentiation, periplakin has a role in the other plakin family members, and BPAG1 [11]. Transfection formation of cornified envelopes, the covalently cross-linked rem- studies have shown that the N-terminus is required for targeting of nants of cells that form a key component of the epidermal barrier periplakin and the dimer of periplakin and envoplakin to mem- [6,7]. Periplakin, together with envoplakin and involucrin, form brane locations [2]. Increased intracellular Calcium concentration has been suggested to play a role in the membrane targeting of

⇑ Corresponding author. Address: School of Biological and Biomedical Sciences, periplakin N-terminus [12] and known binding partners for the Durham University, South Road, Durham, DH1 3LE, United Kingdom. Fax: +44 191 N-terminus such as kazrin and plectin may influence the subcellu- 3341201. lar targeting of the complexes [13,14]. This said, the exact mecha- E-mail address: [email protected] (A. Määttä). nism of the targeting of periplakin to plasma membrane around 1 Present address: Institute of Genetic Medicine, Newcastle University, Interna- desmosomes and the full extent of periplakin interactions at cell tional Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, United Kingdom. border locations has not yet been revealed.

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.057 V. Boczonadi, A. Määttä / FEBS Letters 586 (2012) 3090–3096 3091

Annexins interact with phospholipids providing membrane- been described earlier [14]. 1-D separation of the immunoprecipi- organising functions in processes such as endocytosis, vesicle tated protein complexes was carried out with 4–12% gradient gels transport and membrane targeting [15,16]. The interactions be- (Invitrogen) followed by silver staining using Silver Snap Stain Kit tween annexins and membranes usually require Calcium ions. II (Pierce). Immunoprecipitation with anti-annexin and anti- The membrane-binding domain of annexins has a curved surface periplakin antibodies was carried out in cell-extracts that were that – in the presence of Calcium ions – docks onto phospholipid pre-cleared with protein-G agarose beads by incubating 1–3 ml membranes. Annexin family members have been shown to con- of protein extracts with 10 ll of antibody for 1 h at 4 °C on rocking tribute to epidermal biology. Both annexin A1 and A2 have been platform and then with Protein G beads at 4 °C on rocking plat- detected in epidermis [17]. Annexin A1 forms complexes with form. The immunoprecipitated complexes were washed 3 times S100A11 that is involved in the differentiation pathway induced in lysis buffer for 20 min at 4 °C. Immunoblotting of protein ex- by high Calcium levels. In addition, epidermal growth factor tracts and immunoprecipitated protein complexes and the subse- (EGF) induces cleavage of annexin A1 by cathepsin D at Trp12, quent detection by enhanced chemiluminescence was performed resulting in a loss of interaction with S100A11 [18]. Both annexin as described previously [14,22]. A1 and S100A11 are retained in corneocytes where they get cross-linked to cornified envelopes [19]. 2.4. Subcellular fractioning In this study we show that annexin A9 (also known as pemph- axin), an atypical annexin family member [20,21], co-immunopre- Cells were grown to confluence on 10 cm2 dishes and extracted cipitates with the periplakin N-terminus and co-localises with using saponin as described [22]. Briefly, the first extraction (S1 periplakin both at the lateral cell borders of simple epithelial fraction) was carried out with saponin buffer (0.01% w/v saponin, MCF-7 cells and in differentiating epidermal keratinocytes. 10 mM Tris pH 7.5, 140 mM NaCl, 5 mM EDTA, 2 mM EGTA, 1 mM PMSF (phenyl–methyl sulphonyl fluoride) and protease 2. Materials and methods inhibitor cocktail tablets (Roche) and the insoluble pellet was re- extracted using ice-cold Triton buffer (1% v/v TritonX-100, 2.1. Cell culture and transfections 10 mM Tris pH 7.5, 140 mM NaCl, 5 mM EDTA, 2 mM EGTA, 1 mM PMSF and protease inhibitor cocktail) to generate the S2 Human mammary adenocarcinoma cells (MCF-7) and HaCaT fraction and the Triton-insoluble fraction P fraction. All the sample keratinocytes were maintained in Dulbecco’s minimal essential volumes were adjusted to the same volume with 4Â LSB and equal media (DMEM Sigma, UK) supplemented with 10% foetal calf serum volumes were loaded on a 4–12% bis–Tris gel (Invitrogen) for anal- and 5% GPS (L-glutamine, glucose, penicillin, streptomycin). The ysis by immunoblotting. stably transfected cell lines expressing the pP1/2N construct and control (empty vector) transfected cell lines [14], the annexin A9- 3. Results GFP construct [20], and transient transfections using Lipofectamine [22] have been described earlier. Calcium-induced differentiation We utilised a co-immunoprecipitation approach in a MCF-7 of HaCaT cells was carried as described in [23] with a switch from subclone stably expressing HA-tagged periplakin N-terminus out- a low Calcium (0.06 mM) to high-Calcium (1.6 mM) culture med- lined in our previous publication [14] to identify protein com- ium. To induce a collapse of the intermediate filament , plexes that contain periplakin and influence the subcellular the cells were treated with Okadaic Acid as described [22]. distribution and functions of this cytoskeletal linker protein. The over-expressed periplakin N-terminus is targeted to lateral cell 2.2. Immunofluorescence staining and confocal microscopy membranes of the MCF-7 subclone and shows a dominant negative effect by interfering with cytoskeletal organisation during epithe- Cells were grown on coverslips, fixed and blocked as described lial wound healing [14]. We chose to use simple epithelial cell sys- previously [22] and tissue sections were processed and stained as tem to capture potential interacting partners involved in both described [23]. Tissue samples from periplakin gene targeted mice keratin organisation and epidermal cornified envelope functions [10] and control littermates were obtained from samples collected of periplakin as a use of differentiating keratinocytes would have during the breeding programme for envoplakin/periplakin/involu- resulted in protein cross-linking that complicates proteomic anal- crin triple knock-out mice [7]. The following primary antibodies ysis and is not suitable for a co-immunoprecipitation approach. were used in this study. Periplakin, TD2 and BOCZ1 rabbit polyclo- Previously, we have demonstrated using this approach how plectin nals (1:100 dilution); HA-tag, mouse mAb (1:200, Abcam); keratin interacts with the periplakin N-terminus [14]. To identify the most 8, AE3 mouse mAb (1:100 dilution); annexin A9 (1:200, Abcam) abundant constituents of the protein complexes containing peri- and annexin A1 (1:200, Abcam). Alexa-fluor conjugated secondary plakin and to evaluate the relative amounts of the co-immunopre- antibodies (Invitrogen) were used as described [22]. Stained cells cipitated proteins we carried out 1-dimensional gradient gel were observed Zeiss LSM 510 META confocal microscope imaging electrophoresis of the co-immunoprecipitated proteins followed system equipped with 40X and 63X/1.10 NA lenses. Images were by silver staining. A 36 kDa protein was one the most prominent collected in Multi-Track Mode and composite figures were assem- protein bands in the immunoprecipitated complexes (Fig. 1A). bled in PhotoShop (Adobe). Mass spectrometry of tryptic peptides identified the 36 kDa pro- tein as annexin A9 as it yielded a Mascot score of 176 (protein 2.3. Co-immunoprecipitation and immunoblotting scores >77 considered significant, p < 0.05) (Fig. 1A, B and Supplementary Table 1). Among the other major proteins was ker- Co-immunoprecipitation of periplakin interacting proteins in atin 8. However, we reasoned that any intermediate filament pro- MCF-7C6-Ppl, empty vector transfected control cells and non- teins were likely to be recruited to the complexes by full-length transfected parental MCF-7 cells using Pro-Found Mammalian plectin, which is another previously characterised interacting HA-tag IP/CoIP kit (Pierce); 2-D-electrophoresis using Bio–Rad 2D partner for periplakin [14]. The possibility of plectin bridging gel electrophoresis system; MALDI-TOF separation of tryptic pep- and the periplakin N-terminus together is likely, as the tides (with a Voyager DE-STR, Applied Biosystems, mass spectrom- periplakin N-terminus does not contain the IF-binding domain of eter) and identification of protein using the MASCOT software have the protein. 3092 V. Boczonadi, A. Määttä / FEBS Letters 586 (2012) 3090–3096

Fig. 1. Co-immunoprecipitation of annexin A9 and periplakin. (A) Silver stained 4–12% SDS–PAGE gradient gel of protein complexes immunoprecipitated with anti-HA tag antibodies in stably transfected cell line expressing HA-tagged periplakin N-terminus (Left hand line) or control empty vector transfected cell line (Right hand). The line indicates the HA-tagged periplakin N-terminus and the arrow points a t a 36 kDa protein that was identified as annexin A9. (B) MS spectrum of the tryptic peptides of the annexin A9 protein band. (C) Immunoblotting of the HA-tagged periplakin and annexin A9 in the parental MCF7 cells, Empty vector transfected control cells (EV lane) and in the MCF7-PplN-C6 cell line. (D) Immunoprecipitation with the anti HA-tag of total protein extracts of MCF-7 cells, empty vector transfected cells and the MCF7-PplN-C6 cells followed by immunoblotting with the anti-HA antibody for the periplakin N-terminus (top panel) or anti-annexin A9 antibody (Bottom panel). (E) Immunoprecipitation of the MCF7 cells with anti-annexin A9 antibody or protein-G Sepharose beads only (CTRL lane) followed by immunoblotting with anti-periplakin antibody (BOCZ1). Line in the right indicates migration of 170 kDa Molecular Weight marker.

The presence of annexin A9 in the immunoprecipitated protein itated the endogenous 195 kDa periplakin in the MCF-7 cells complexes was confirmed by immunoblotting (Fig. 1C). Both the (Fig. 1E). HA-tagged periplakin N-terminus and annexin A9 were detected The interaction between HA-tagged periplakin N-terminus and in complexes immunoprecipitated with an anti-HA antibody from annexin A9 is supported by their co-localisation of at cell borders the MCF7-C6 cell line whereas no annexin A9 was detected when in confluent MCF-7 C6-Ppl cells (Fig. 2 A–C). Likewise, the endog- either an empty vector transfected control cell line or the parental enous full length periplakin co-localises with annexin A9 at the MCF-7 cell line were used for the immunoprecipitation. Notably, cell borders in untransfected MCF-7 cells (Fig. 2 D–F), where an- annexin A9 was expressed by all the three cell lines (Fig. 1D). This nexin A9 occupies the lateral membranes to the same extent as confirms that annexin A9 does not interact non-specifically with periplakin (Inserts in Fig. 2 D–F) as opposed to a restricted distri- HA-antibody conjugated beads used in the IP assays. Finally, we bution of other junctional proteins, and beta- utilised co-immunoprecipitation of the endogenous proteins to [22]. Finally GFP-tagged annexin A9 co-localised with HA-tagged demonstrate that the annexin–periplakin complexes can be found periplakin at cell–cell borders of the MCF-7 clone stably transfec- in untransfected cells as annexin A9 antibodies co-immunoprecip- ted with the periplakin construct (Fig. 2 G–I). No other annexins V. Boczonadi, A. Määttä / FEBS Letters 586 (2012) 3090–3096 3093

Fig. 2. Co-localisation of periplakin and annexin A9 at lateral cell membranes of MCF7 cells. (A) HA-tagged periplakin N-terminus (green channel) and (B) annexin A9 (red channel) co-localise at cell borders of the stably transfected MCF7-PplN-C6 cell line (C). Merged panel of A and B. (D) Endogenous full-length periplakin (green channel) and (E) annexin A9 (red channel) co-localise at cell borders of the parental MCF7 cell line. Inserts in the bottom left hand corners show X–Z confocal projection of a cell border. (F) Merged image. (G) Transiently transfected annexin A9-GFP co-localises with (H) HA-tagged periplakin N-terminus (red channel). (I) Merged panel. (J) Annexin A1 does not localise to lateral plasma membrane with (K) annexin A9 in MCF7 cells. (L) Merged panel. (M) AnnexinA9-GFP construct transfected into control MCF7 cells. The arrowhead indicates localisation at the cell border between the two GFP positive cells. (N) Trasfection of GFP alone into control MCF-7 cells. Scale bar corresponds to 20 lm in all panels. 3094 V. Boczonadi, A. Määttä / FEBS Letters 586 (2012) 3090–3096 co-immunoprecipitated with the periplakin N-terminus and, for lar fractionation, so that the protein is more abundant in the solu- example, annexin A1 does not show similar strong lateral mem- ble cytoplasmic fraction (Fig. 3G). This is in accordance with our brane targeting as annexin A9 in MCF-7 cells (Fig. 2 J–L). In further previously published results that show increased solubility of peri- control experiments, GFP-annexin A9 was also targeted to cell plakin after OA treatments [22]. The increase presence of keratin 8 borders in control MCF-7 cells (Fig. 2M) indicating that the local- in the soluble pool (Fig. 3 G) acts as a positive control for the OA isation of the GFP fusion protein was not dependent on the over- treatment. expression of the periplakin N-terminus, whereas GFP alone was One of the major functional roles of periplakin is the formation evenly distributed in the cells without any non-specific cell border of the initial sub-plasma membrane scaffold for the cornified cell localisation (Fig. 2N). envelope assembly during the acquisition of the epidermal barrier. Further evidence for a co-localisation of annexin A9 and peri- Likewise several annexin family members have previously been plakin in the same protein complexes was obtained by studying implicated in the differentiation of the epidermal keratinocytes. changes in the subcellular localisation of these proteins after a dis- Therefore we next investigated the localisation of annexin A9 ruption of the intermediate filament cytoskeleton by Okadaic acid expression in skin. We found that annexin A9 co-localises with (OA). Inhibition of protein phosphatases by OA results in hyperph- periplakin in both interfollicular epidermis and in hair follicles of oshorylation of intermediate filament proteins leading to a both newborn and adult mouse skin (Fig. 4 A). In the interfollicular disassembly of the filament network. We have previously demon- epidermis the most striking co-localisation was seen at the cell strated how the periplakin C-terminus mediates redistribution of borders of differentiating, flattened keratinocytes. Prominent the protein to clusters with the collapsing intermediate filaments co-localisation was also seen in the hair follicle (Fig. 4 A). As the [22]. Thus, we wanted to establish, whether periplakin and annex- staining intensity of annexin A9 seemed to increase upon keratino- in A9 stay together in the same protein complex when collapsing cyte differentiation, we investigated the corresponding protein lev- the intermediate filament cytoskeleton alters the localisation of els in cultured HaCaT keratinocytes following Calcium-induced periplakin. A 2-h long treatment of MCF7 cells results in formation differentiation. The expression level of annexin A9 was markedly of keratin8/18 clusters that contain periplakin (Fig. 3 A–C). Like- increased within 24 h of switch to High Calcium culture medium wise, annexin A9 and periplakin co-localise in cytoplasmic aggre- (Fig. 4 B) indicating either increased annexin A9 gates in OA treated cells (Fig. 3 D–F). Furthermore, OA-treatment or protein stability in differentiating keratinocytes. Intriguingly, causes a change in the distribution of annexin A9 during subcellu- also the subcellular distribution of the protein was altered. In

Fig. 3. Co-localisation of periplakin and annexin A9 in Okadaic acid–treated cells. (A) Keratin 8 (red channel) and (B) periplakin (green channel) co-localise in cytoplasmic aggregates in MCF7 cell treated for 2 h with Okadaic acid. (C) Merged panel. (D) Cytoplasmic aggregates of periplakin (red channel) and (E) annexin A9 in OA treated cells. (F) Merged panel. (G) Okadaic-acid treatment re-distributes all annexin A9 into the saponin-soluble cytoplasmic fraction. Keratin 8 immunoblot was used as a control for OA treatment and fractionation. V. Boczonadi, A. Määttä / FEBS Letters 586 (2012) 3090–3096 3095

Fig. 4. Annexin A9 expression in epidermal keratinocytes. (A) annexin A9 (green channel) and periplakin (red channel) immunofluorescence in adult and newborn mouse skin. Dapi staining was used to visualise the nuclei. The stratum corneum staining in the Newborn mouse skin panel is due to non-specific binding of the secondary antibody. Inserts in the bottom corners of annexin A, periplakin and merged panels show higher magnification of the epidermal staining. (B) Annexin A9 immunoblotting of HaCaT cells induced to differentiate by a switch to high Calcium culture media for the indicated times. Keratin blotting (Pan-K) shows protein loading. Relative annexin A9 expression levels compared to the low Calcium culture and normalised with the keratin expression are shown below. (C) Subcellular distribution of annexin A9 in HaCaT Cells grown in either high or low calcium culture media. immunoblot was used as a control for the fractionation. S1, cytosolic fraction; S2, Triton soluble fraction; P3 Triton- insoluble fraction. (D). Annexin A9 expression in control (Left) and periplakin knock-out (Right) mouse skin. Inserts in the bottom right hand corners show epidermis in higher magnification and the white dotted line indicates the dermo-epidermal junction. low calcium conditions the majority of annexin A9 is readily ex- said, the results do not exclude the possibility that other, uncharac- tracted from the cytoplasm of HaCaT cells whereas after the induc- terised proteins present in the complexes mediate the interaction. tion of the differentiation annexin A9 is mostly localised in the The co-localisation with annexin-A9 reveals an additional layer Triton-insoluble membrane and cytoskeletal fraction (Fig. 4 C). Un- of complexity of periplakin interactions at cell borders in simple like after OA-treatment, this change in subcellular distribution is and stratified epithelia. Previously, it has been shown to participate not linked to altered solubility of intermediate filaments as keratin in the initial scaffold for cornified envelope assembly together with 14 remains in insoluble cytoskeletal pool in both low and high Cal- envoplakin and involucrin [1–3,6–8]. However, the periplakin N- cium culture conditions. However, the expression of annexin A9 in terminus appears to have several possible direct and indirect inter- mouse epidermis does not require periplakin as the expression le- action partners. Kazrins, a group of at least five alternatively vel and distribution of annexin A9 was not altered in periplakin- spliced isoforms that participate in desmosome assembly and epi- ablated adult mouse backskin (Fig. 4 D). dermal differentiation also bind periplakin N-terminus but can be targeted to at least some subcellular locations (such as acetylated 4. Discussion ) independently on periplakin [13,24,25]. Plectin, a large multifunctional cytolinker protein can also be found in pro- We demonstrate in this paper that annexin A9 is found in pro- tein complexes containing periplakin and both of these plakin pro- tein complexes containing periplakin, a cytoskeletal linker protein teins influence keratin organisation and cell migration [14]. Taken belonging to the plakin family. We identified annexin A9 in a pro- together, these previous findings and the novel interaction de- teomic screen of protein complexes co-immunoprecipitated from scribed here indicate that the protein complexes involved in the MCF-7 cells stably transfected with HA-tagged periplakin N-termi- initial assembly of cornified envelopes are likely to involve more nus. The interaction was confirmed by co-immunoprecipitation of than the three initially suggested components. full-length endogenous proteins and by co-localisation of the pro- Interestingly, both periplakin and annexin A9 are targets of teins at cell borders of MCF-7 cells and in mouse epidermis. This autoantibodies in epidermal blistering diseases. The linker domain 3096 V. Boczonadi, A. Määttä / FEBS Letters 586 (2012) 3090–3096 of periplakin (and other plakin family member) is recognised by [3] Kazerounian, S., Uitto, J. and Aho, S. (2002) Unique role for the periplakin tail in autoantibodies in paraneoplastic pemphigus 5,6] whereas annexin intermediate filament association: specific binding to keratin 8 and . Exp. Dermatol. 11, 428–438. A9 is one of several targets in addition to well-characterised [4] Kiyokawa, C., Ruhrberg, C., Nie, Z., Karashima, T., Mori, O., Nishikawa, T., Green, desmoglein autoantibodies in Pemphigus Vulgaris [21]. K.J., Anhalt, G.J., DiColandrea, T., Watt, F.M. and Hashimoto, T. (1998) Previous studies on annexin A9 have indicated that it differs Envoplakin and periplakin are components of the paraneoplastic pemphigus antigen complex. J. Invest. Dermatol. 111, 1236–1238. from other annexin family members in its response to Calcium. [5] Mahoney, M.G., Aho, S., Uitto, J. and Stanley, J.R. (1998) The members of the At 1 mM Calcium concentration where the other annexins show plakin family of proteins recognized by paraneoplastic pemphigus antibodies a complete or near complete association with liposomes, annexin- include periplakin. J. Invest. Dermatol. 111, 308–313. [6] Kalinin, A.E., Kajava, A.V. and Steinert, P.M. (2002) Epithelial barrier function: A9 demonstrated only a negligible binding. Only at 2.5 mM Ca did assembly and structural features of the cornified envelope. Bioessays 24, 789– annexin A9 co-pellet with liposomes, but the interaction was not 800. reversible upon chelation of calcium but the release required addi- [7] Sevilla, L.M., Nachat, R., Groot, K.R., Klement, J.F., Uitto, J., Djian, P., Määttä, A. and Watt, F.M. (2007) Mice deficient in involucrin, envoplakin, and periplakin tion of triton X100 to the complexes [20]. Furthermore unlike its have a defective epidermal barrier. J. Cell Biol. 179, 1599–1612. closest relative, annexin A2, ANXA9 was not able to bind [8] Steinert, P.M. and Marekov, L.N. (1999) Initiation and assembly of the cell S100A10 in overlay assays [20]. It would be interesting to investi- envelope barrier structure of stratified squamous epithelia. Mol. Biol. Cell 10, gate whether annexin A9 and periplakin are preferentially targeted 4247–4261. [9] Marekov, L.N. and Steinert, P.M. 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(2004) Kazrin, a novel periplakin-interacting protein associated with desmosomes and the pears that annexin A9 is targeted to membrane locations indepen- keratinocyte plasma membrane. J. Cell Biol. 166, 653–659. dently of periplakin. This can, importantly, provide a mechanism [14] Boczonadi, V., McInroy, L. and Määttä, A. (2007) Cytolinker cross-talk: for the initial assembly of the cornified envelopes. So far, none of Periplakin N-terminus interacts with plectin to regulate keratin organisation the previously identified periplakin interactions does explain, and epithelial migration. Exp. Cell Res. 313. 3579-359. [15] Gerke, V., Creutz, C.E. and Moss, S.E. (2005) Annexins: Linking Ca2+ signalling how the periplakin-dependent scaffold is assembled at the plasma to membrane dynamics. Nat. Rev. Mol. Cell Biol. 6, 449–461. membrane surrounding and between desmosomes. Further exper- [16] Monastyrskaya, K., Babiychuk, E.B. and Draeger, A. (2009) The annexins: iments, for example generation of annexin A9 null mice will ad- spatial and temporal co-ordination of signaling events during cellular stress. Cell. Mol. Life Sci. 66, 2623–2642. dress the question whether annexin A9 has a direct and unique [17] Ma, A.S. and Ozers, L.J. (1996) Annexins I and II show differences in subcellular role in targeting or tethering periplakin to plasma membrane. localization and differentiation-related changes in human epidermal keratinocytes. Arch. Dermatol. Res. 288, 596–603. [18] Sakaguchi, M., Murata, H., Sonegawa, H., Sakaguchi, Y., Futami, J., Kitazoe, M., Acknowledgements Yamada, H. and Huh, N.H. (2007) Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic We would like to thank Professor Volker Gerke (University of phospholipase A2 in normal and malignant squamous epithelial cells. J. Biol. Chem. 282, 35679–35686. 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