Caspase 6 Disconnects Intermediate Filament-Binding Domain of Periplakin from Its Actin-Binding N-Terminal Region

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Breaking the Connection: Caspase 6 Disconnects Intermediate Filament-Binding Domain of Periplakin from its Actin-Binding N-Terminal Region Andrey E. Kalinin,Ã Alexandr E. Kalinin,Ã Mikko Aho,w Jouni Uitto,w and Sirpa Ahow ÃLaboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA; wDepartment of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Periplakin is a member of the plakin family of cytolinkers that connect cytoskeletal networks to each other as well as to the cell junctional complexes. Here, we demonstrate a direct molecular interaction between actin and periplakin. Furthermore, the oligomerization state of periplakin was shown to determine specificity of its binding to intermediate filaments (IF) in vitro. Both the filament association and the cell membrane localization of periplakin were confirmed in the cells overexpressing human periplakin. Double labeling of the N- and C-terminally tagged periplakin revealed unexpected lack of co-localization of periplakin ends in a confluent culture, and separation of the periplakin ends was even more pronounced in apoptotic cells. Western analysis revealed that after induction of apoptosis, periplakin becomes cleaved close to its C-terminal tail. Only the distinct cleavage products, but not the full-length periplakin, were present in the cells detached from the solid support during the apoptotic process. We show that caspase 6 cleaves periplakin at an unconventional recognition site, amino acid sequence TVAD. Thus, the separation of periplakin ends disconnects the actin-binding head-rod domain from the IF-binding C-terminal domain. We show that specific cleavage products co-exist with the full-length periplakin in cells, suggesting physiological consequences due to their altered binding specificities. Key words: actin/apoptosis/caspases/intermediate filaments/periplakin J Invest Dermatol 124:46 –55, 2005 Periplakin is one of the five plakin proteins that share a microfilaments. Plectin also binds a6b4 integrin through its common structure of a central rod domain flanked by N- N-terminal domain (Rezniczek et al, 1998; Geerts et al, and C-terminal globular domains; the other members of this 1999; Litjens et al, 2003). Periplakin has been demonstrated family of proteins are desmoplakin, plectin, envoplakin, and to co-localize with cortical actin at the plasma membrane of the 230-kDa bullous pemphigoid antigen (BPAG1) (for re- cultured keratinocytes (DiColandrea et al, 2000). On the views, see Ruhrberg and Watt, 1997; Fuchs and Yang, other hand, the ability to bind intermediate filaments (IF) 1999; Leung et al, 2001a; Leung et al, 2002; Huber, 2003). through the C-terminal region is shared among plakins (Le- The central rod domain is composed of heptad repeats ung et al, 2002; Huber, 2003). The length of the C-terminal potentially forming a coiled-coil structure that assembles globular domain varies from about 2000 residues in plectin plakin polypeptides into parallel homo- or heterodimers to just 111 residues in periplakin. Although IF binding is (Kalinin et al, 2004). The N-terminal domain displays highest mediated through this C-terminal region, the adjacent rod structural homology between five plakin proteins. Function- domain may also contain sequences critical for these inter- ally, the head domain of desmoplakin is involved in binding actions (Stappenbeck and Green, 1992; Nikolic et al, 1996; to the desmosomal plaque proteins (Bornslaeger et al, DiColandrea et al, 2000; Choi et al, 2002; Karashima and 1996) and it also binds to actin (Huen et al, 2002). Alterna- Watt, 2002; Kazerounian et al, 2002; van den Heuvel et al, tive splicing of exons encoding the actin-binding domain 2002; Fontao et al, 2003). The ability to bind actin micro- generates specific isoforms of BPAG1 (Leung et al, 2001b) filaments via its N-terminal domain and to provide a con- and plectin (Fuchs et al, 1999; Andra¨ et al, 2003; Garcia- nection to the IF via the C-terminal domain has designated Alvarez et al, 2003), with unique binding properties to actin plectin as a prototype molecule of these versatile cytolinkers (Steinbo¨ ck and Wiche, 1999). Periplakin, although smaller than plectin, also emerges as a potential cytolinker. Abbreviations: BSA, bovine serum albumin; IF, intermediate fila- Periplakin was originally identified as a precursor of the ments; IIF, immunofluorescence; FLAG, synthetic epitope tag; HA, cornified cell envelope in terminally differentiated epidermal epitope tag derived from influenza virus hemagglutin protein; keratinocytes (Simon and Green, 1984; Ma and Sun, 1986; MDCK, Madin–Darby canine kidney; PVDF, polyvinylidene fluoride; Ruhrberg et al, 1997), but its expression has also been RT, room temperature; SDS-PAGE, sodium dodecyl sulfate-polya- crylamide gel electrophoresis single letter code for amino acid demonstrated in other tissues with prominent epithelial residues: T, threonine; V, valine; A, alanine; D, aspartic acid component (Aho et al, 1998; Kazerounian et al, 2002). Un- Copyright r 2004 by The Society for Investigative Dermatology, Inc. 46 124 : 1 JANUARY 2005 CASPASE CLEAVAGE OF PERIPLAKIN CYTOLINKER 47 expected protein–protein interactions, identified through the dressed previously. Because both the full-length periplakin yeast two-hybrid screening, indicate novel functions for and its N- and C-terminal cleavage products may simulta- periplakin (Aho and Kazerounian, 2003). Such periplakin neously be present in the cells and tissues, the appearance interaction partners include the intracellular domain of the of periplakin signal is dependent on the tools and methods hemidesmosomal transmembrane protein-type XVII colla- used for their detection. Here, we provide evidence of direct gen/BPAG2/BP180 (Aho et al, 1998; Aho, 2004a), a G-pro- selective interaction of periplakin C-terminus with individual tein receptor in the brain (Feng et al, 2003), protein kinase B/ IF proteins and assembled IF, as well as show direct mo- c-Akt involved in regulation of apoptosis (van den lecular interaction between filamentous actin and periplakin Heuvel et al, 2002), the mannose-6-phosphate receptor head and rod domains in vitro. We use N- and C-terminally (MPR300) involved in intracellular protein trafficking (Storch, tagged periplakin to demonstrate that cleavage of peripl- 1999), and the high-affinity immunoglobulin G (IgG) receptor akin by caspase 6 produces two fragments, selectively FcgRI in myeloid cells, enhancing its capacity to bind, in- abolishing protein–protein interactions, and resulting in the ternalize, and present antigens on major histocombatibility separation of the terminal domains in living cells. complex II (Beekman et al, 2004a,b). The yeast two-hybrid screening for periplakin interaction partners also identified a novel protein, periphilin, which is present at cell membranes Results in terminally differentiated keratinocytes (Kazerounian and Specific interaction of periplakin with IF proteins Previ- Aho, 2003). In addition, this technology has confirmed a ous studies have described filamentous distribution for pe- specific binding of periplakin to keratin 8 and vimentin riplakin C-terminal domain in epithelial cells transfected with (Kazerounian et al, 2002; van den Heuvel et al, 2002). Based various expression constructs (DiColandrea et al, 2000; on the established tissue distribution and the subcellular Karashima and Watt, 2002; Kazerounian et al, 2002), and localization of periplakin, the physiological relevance of the direct interaction with IF extracted from cultured cells has two-hybrid interactions has remained unclear. A novel also been reported (Karashima and Watt, 2002). We and monoclonal antibody recognizing an epitope close to the others (Kazerounian et al, 2002; van den Heuvel et al, 2002) C-terminal end of periplakin rod domain, however, revealed also demonstrated in yeast two-hybrid assay a specific in- an unexpected subcellular localization and tissue distribu- teraction of periplakin C-terminal domain with keratin 8 and tion pattern for this protein (Aho, 2004a). An increasing vimentin. As there are many more individual IF proteins (for number of protein–protein interactions suggests that pe- a review, see Parry and Steinert, 1999), which are specif- riplakin C-terminus may be masked and not accessible for ically expressed in cells and tissues where periplakin is the C-terminus-specific antibodies, which are widely used known to be abundant, it was of interest to investigate the for detection of periplakin. There is also a possibility that capability of periplakin to bind to other IF proteins as well. due to proteolytic processing, certain protein domains are The capacity of full-length periplakin or its C-terminal not present in cells and tissues. fragments (Fig 1) to bind bacterially expressed purified in- Caspases are aspartate-specific cysteine proteases that dividual IF proteins (Fig 2a) was analyzed by the dot-blot become activated and cleave cellular proteins, mediating overlay assay (Fig 2b). Full-length periplakin exhibited elimination of damaged cells during apoptosis and contrib- strong binding to keratins 8 and 14 (Fig 2b, panel P). Weak- uting to programmed cell death during development er binding was also observed with vimentin and keratins 1 (Jacobson et al, 1997; Nicholson, 1999; Zeuner

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