Annexin A9 Is a Periplakin Interacting Partner in Membrane-Targeted Cytoskeletal Linker Protein Complexes ⇑ Veronika Boczonadi 1, Arto Määttä
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 586 (2012) 3090–3096 journal homepage: www.FEBSLetters.org Annexin A9 is a periplakin interacting partner in membrane-targeted cytoskeletal linker protein 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 keratin 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 proteins 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 actin 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 gene 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, envoplakin 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, plectin 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 cytoskeleton, 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