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Breaking the Connection: Caspase 6 Disconnects -Binding Domain of Periplakin from its -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 pe- riplakin. 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 that share a microfilaments. 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 , plectin, , 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 cytolin- kers (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 ; 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 8 and 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 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 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 et al, and 5. Although periplakin C-terminal fragment, containing 1999; Adams, 2003; Lawen, 2003; Schwerk and Schulze- both the C-terminal third of the rod and the entire tail do- Osthoff, 2003). Characteristic morphological changes main (Fig 2b, panel Pr231t), showed similar binding pattern during apoptosis are due to the cleavage of intracellular as the full-length periplakin, further shortening of the rod proteins, especially those responsible for the cell–cell or domain (Fig 2b, panel Pr89t) largely abolished the binding to cell–matrix adhesion, as well as the integrity of the nuclear membrane and the IF network. Desmosomal proteins, in- cluding desmoplakin, are coordinately cleaved during apoptosis primarily through the action of caspase 3 (We- iske et al, 2001), whereas plectin has been reported to function as an early substrate for caspase 8 (Stegh et al, 2000; Beil et al, 2002). Destruction of the nuclear lamina and the collapse of the nucleus are mediated through caspase 6 cleavage of nuclear (Orth et al, 1996; Ruchaud et al, 2002). Caspase 6 is also responsible for cleavage of (Caulin et al, 1997), keratins 15 and 17 (Badock et al, 2001), vimentin (Byun et al, 2001), and (Chen et al, 2003), thus systematically targeting the IF network during apoptosis. We recently reported that in HaCaT keratin- ocytes, periplakin is coordinately cleaved with other plakin Figure 1 proteins, and in vitro periplakin is preferentially cleaved by Periplakin constructs. Periplakin constructs used in in vitro binding caspase 6 (Aho, 2004a,b). assays. Periplakin amino acid residues encoded by each construct are indicated, and the ability of each domain to bind intermediate filaments The specific cleavage of periplakin and the cleavage (IF) and F-actin (further detailed in Figs 2 and 4, respectively) is sum- products present in cells and tissues have not been ad- marized on the right. 48 KALININ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2 C-terminal region of periplakin is responsible for selective binding to intermediate fila- ments (IF). (a) Coomassie Blue-stained gel showing purified IF proteins, which were used in the dot-blot overlay assay (b) and co-sedi- mentation studies (c–e). (b) IF proteins (1 mg each) were immobilized on polyvinylidene fluo- ride membrane, which was probed with purified periplakin fragments indicated on the left. Bound periplakin was detected by antibodies as de- scribed in Materials and Methods. No periplakin was added in the control experiment. Equal loading of IF proteins was verified by Amido Black staining of the membrane. (c) The full- length periplakin or its deletion constructs, as indicated on the top of each panel, were incu- bated with or without IF assembled from keratins 8 and 18 (K8 and K18). Supernatants (s) and pellets (p) after ultracentrifugation were analyzed by gel electrophoresis. The arrowhead marks the position of each protein tested for binding. (d) Quantitative analysis of Pr231t binding to K8/18 IF. Amount of protein bound to the IF was deter- mined by densitometry of the Coomassie Blue- stained gels. Results of three independent experiments are shown. Error bars represent standard deviation. (e) Co-sedimentation assay with IF assembled from K5 and K14.

keratin 8 and vimentin. Accordingly, the periplakin tail do- main alone (Fig 2b, panel Pt) was not able to bind to keratin 8 or vimentin. Co-sedimentation assays, measuring the ability of pe- riplakin and its domains to bind to IF, confirmed that the periplakin tail domain is necessary but not sufficient to bind IF assembled from keratins 8 and 18. On the one hand, proteins Prt and Pr231t, but not corresponding fragments Pr and Pr231 without the tail domain, were detected in the pellet fraction after the incubation with the IF (Fig 2c, panels Prt and Pr231t vs panels Pr and Pr231). On the other hand, constructs containing the tail domain, preceded by just 89 Figure 3 residues of the rod domain, or the tail domain alone did not Substantial part of the rod domain is required for oligomerization show significant binding (Fig 2c, panel Pr89t, and data not of the periplakin in vitro. (a) Purified recombinant proteins were chemically cross-linked (lanes ‘‘ þ ’’) before separation through sodium shown). In addition, the periplakin head domain did not bind dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie the IF (Fig 2c, panel Ph). Similar results were obtained when Blue staining. Control samples were incubated without cross-linker vimentin IF were used in the co-sedimentation assay, ex- (lanes ‘‘’’). Positions of molecular mass standards (kDa) are indicated on the left. (b) Apparent molecular mass of the recombinant proteins cept that the binding capacity of vimentin IF for periplakin (open circles) was estimated by size exclusion chromatography. was somewhat lower than that of keratin 8/18 IF (data not shown). In case of keratin 8/18 IF, binding was saturated at a stoichiometry of one Pr231t polypeptide chain per keratin tetramer as calculated from Scatchard analysis. The Kd, the one hand, the major product of chemical cross-linking of estimated from these binding data, is 2.11 mM (Fig 2d). Pr231t was an oligomer with an apparent molecular mass of Surprisingly, in contrast to the overlay assay that demon- about 140 kDa as estimated by sodium dodecyl sulfate- strated a rather strong interaction between periplakin C- polyacrylamide gel electrophoresis (SDS-PAGE) (Fig 3a, terminal fragments with individual , the co-sedi- panel Pr231t), whereas the majority of Pr89t remained in its mentation experiments failed to demonstrate any significant monomeric form (Fig 3a, panel Pr89t). On the other hand, binding to IF assembled from keratins 5 and 14 (Fig 2e, and the molecular mass of Pr89t was estimated as 28 kDa by data not shown). size exclusion chromatography (Fig 3b), which is reasonably Differences in IF-binding properties between constructs close to 23.4 kDa calculated from its amino acid sequence. Pr231t and Pr89t could be due to their different oligomeric On the same column, Pr231t appeared as a molecule with state. To address this possibility, we used cross-linking ex- an apparent mass of about 230 kDa, which is several fold periments and size exclusion chromatography (Fig 3). On higher than 41.6 kDa calculated for its monomer. We 124 : 1 JANUARY 2005 CASPASE CLEAVAGE OF PERIPLAKIN CYTOLINKER 49 conclude that Pr231t is forming oligomeric complexes, per- haps as a result of lateral association of rod domain se- quences present only in the Pr231t protein. The subcellular distribution of periplakin was studied in cultured cells using double labeling for the N- and C-termi- nal tags (Fig 5a, b). In sparse cultures, where cells are well spread and take a large surface area, the signals co-local- ized and were detected in the cytoplasm in a filamentous pattern. This result supports the association of periplakin with IF, especially in non-confluent cells, which have not yet established strong cell–cell adhesion. The filamentous pat- tern is in agreement with the previously reported IF asso- ciation of overexpressed periplakin (DiColandrea et al, 2000; Karashima and Watt, 2002; Kazerounian et al, 2002).

Direct molecular interaction between periplakin and F- actin Although periplakin has been shown to co-localize and co-distribute with cortical actin (DiColandrea et al, 2000), the possibility of direct molecular interaction of pe- riplakin with filamentous actin has not been explored. Therefore, the actin-binding activity of each periplakin frag- ment was assessed through co-sedimentation with F-actin. Full-length recombinant periplakin exhibited weak but de- tectable affinity to actin filaments in vitro (Fig 4a). The N- terminal head domain showed similar binding properties, whereas the periplakin rod domain, surprisingly, showed even stronger binding to F-actin. None of the C-terminal periplakin fragments showed co-sedimentation with actin (Fig 4a, panel Pr231t, and data not shown). When a range of protein concentrations was used to determine the actin- binding parameters, an apparent Kd of about 6 mM was obtained for Ph (Fig 4b). This value is several fold higher than dissociation constants estimated for actin-binding do- mains of other plakins: BPAG1 (Kd ¼ 0.21 mM) (Yang et al, 1996) and -actin cross-linking factor (MACF) (Kd ¼ 0.35 mM) (Karakesisoglou et al, 2000). Therefore, bind- ing of Ph to actin can be described as weak, although spe- cific. On the other hand, binding saturation for Pr was not Figure 4 The head and rod domains of periplakin bind to filamentous actin. obtained even at concentrations as high as 8 mM, indicating (a) F-actin co-sedimentation assay was performed in a manner similar a stronger but unspecific character of the binding (Fig 4c). to intermediate filament-binding experiments. The arrowhead marks In Madin–Darby canine kidney (MDCK) cells grown to the position of each protein tested for binding. For the actin binding, a confluency, the double-tagged periplakin appeared at the positive (a-actinin) and negative (bovine serum albumin (BSA)) controls are shown. (b and c) Quantitative analysis of Ph and Pr domains binding periphery of the cells along the cell–cell junctions (Fig 5c), to F-actin, respectively. Results of three independent experiments are which is in contrast to the filamentous appearance seen in shown. Error bars represent standard deviation. Note saturation of sparse cultures (Fig 5b). The staining pattern in the conflu- binding with increasing concentrations of Ph and absence of the sat- ent culture suggests co-localization of periplakin with cor- uration in case of Pr. tical actin as was shown previously (DiColandrea et al, MDCK cells expressing double-tagged periplakin and mon- 2000). Furthermore, in some cells FLAG-tag signal was itored both tags through immunofluorescence (IIF) staining. barely detectable, whereas a much brighter HA-tag signal After induction of apoptosis by staurosporine (STS), shrink- was seen as diffuse cytoplasmic staining. age of the cells was detectable after 1-h treatment (data not shown) and after 3-h treatment diamidinophenyl indole Induction of apoptosis results in separation of peripl- (DAPI) staining revealed prominent DNA condensation and akin termini The surprising lack of co-localization of the nuclear fragmentation (Fig 5d). In apoptotic cells, the signals two signals in confluent cultures could be either due to of FLAG- and HA-tags did not co-localize. The N-terminal epitope masking, as was suggested previously (Aho, end of periplakin was detected mainly in aggregates, 2004a), or a result of a proteolytic cleavage of periplakin. whereas the C-terminal end appeared in the cytoplasmic We recently found that caspase 6 cleaves periplakin in vitro staining pattern, indicating the separation of the two ends (Aho, 2004a), and in HaCaT keratinocytes after induction of in the remaining adherent cells and cell debris. Periplakin apoptosis, the endogenous periplakin is cleaved coordi- cleavage might also occur during cell/culture maturation, nately with other plakin proteins (Aho, 2004b). To track the thus explaining the separation of the two signals observed fate of periplakin ends in apoptotic cells, we again used in confluent cultures (Fig 5c). 50 KALININ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 5 Subcellular distribution of periplakin depends on the physiological state of the cells. (a)A construct, encoding double-tagged periplakin with an N-terminal FLAG- and a C-terminal HA- epitope tag, was expressed in Madin–Darby ca- nine kidney (MDCK) cells. In addition to the tag-specific antibodies, periplakin is recognized by the monoclonal antibody PPL-488, specific for aa 1548–1583. Periplakin domains responsible for F-actin and intermediate filament (IF) binding, and the caspase 6 cleavage site are indicated. N-terminally FLAG-tagged and C-terminally HA- tagged periplakin overexpressed in MDCK cells was double-labeled with antibodies against the FLAG-epitope (red) and the HA-epitope (green), and the nuclei were visualized through diami- dinophenyl indole (DAPI) staining (blue). (b) The filamentous staining pattern of both tags in growing cells suggests IF association for full- length periplakin (arrow). (c) In confluent cultures, periplakin localized along the cell plasma mem- brane (arrow). An unexpected lack of co-locali- zation of the signals is evident in some cells. (d) Separation of periplakin domains after induc- tion of apoptosis. Distinct aggregates of the N-terminal part of the periplakin were detected through the FLAG-tag signal, especially in the cells with fragmented or condensed nuclei, but these aggregates did not stain with the HA-tag antibody (arrows). Another type of staining, where the FLAG-tag antibody gave a dim signal and the HA-tag antibody was seen throughout the cytoplasm, is indicated by the arrowheads. In an occasional cell both signals co-localized, ei- ther throughout the cytoplasm (encircled with a solid line), or as prominent aggregates (encircled with a broken line). Scale bar:25mm.

The examination of cell cultures revealed that after STS experiment was repeated using a caspase 6-specific inhib- addition, cells became rounded, lost their contact with the itor zVEID-fmk (data not shown). This inhibitor clearly re- matrix, and were gradually released into the culture medi- duced apoptosis and prevented periplakin cleavage in um. Western analysis was used to further examine the fate adherent cells, although it did not completely prevent cells of periplakin and its fragments both in adherent and de- from detaching, and in the floating cell pool we detected tached cells (Fig 6). In adherent cells, a small amount of the both full-length periplakin and the cleavage fragments, sug- N-terminal cleavage product, corresponding to the head- gesting that the cleavage of overexpressed periplakin is not rod domain of periplakin (Fig 6), was present even in the a prerequisite for the loss of adhesion of MDCK cells. These vehicle-treated cells (0-time). Significant increase of both results also indicate that there may be other caspases, be- the N- and the C-terminal cleavage products was detected sides caspase 6, cleaving periplakin. in a time-dependent manner after addition of STS, although the full-length periplakin constituted the major fraction of Caspase 6 cleaves periplakin at an unconventional periplakin in adherent cells even at 3-h time point. Signif- cleavage site Recombinant caspases 1–10 were tested icantly, only the cleavage products, but not the full-length in vitro for their ability to produce distinct fragments from periplakin, were present in the detached cells. Based on the the tagged periplakin in the MDCK cell extract (Fig 7), and intensity of actin signal (Fig 6, right panel), the inhibition of caspase 14 was tested on the full-length recombinant pe- caspase activity by zVAD-fmk (Biomol, Plymouth Meeting, riplakin (Kalinin, unpublished). Only caspase 6 cleaved Pennsylvania) greatly reduced the number of detached periplakin, producing a fragment of about 180 kDa. cells. Moreover, the caspase inhibitor almost quantitatively Furthermore, the HA-tag-specific antibody revealed a dis- eliminated the periplakin cleavage product from the adher- tinct fragment, approximately 15–20 kDa in size, similar to ent cells, suggesting the presence of some caspase activity the periplakin cleavage product, described in Fig 6. These in these cells already before STS treatment. results confirmed the previous work, where the utilization of As we showed earlier, the periplakin could serve as a glutathione S-transferase-fusion proteins narrowed the ca- substrate for caspase 6 in vitro (Aho, 2004a). Therefore, the spase cleavage site to aa 1584–1645 (Aho, 2004a). This 124 : 1 JANUARY 2005 CASPASE CLEAVAGE OF PERIPLAKIN CYTOLINKER 51

Figure 8 Determination of caspase 6 cleavage site. (a) Purified recombinant protein Pr231t (lane 1) was cleaved by caspase 6 into two fragments (lane 2). His-tagged C-terminal fragment (lane 3) was purified. (b) Amino Figure 6 acid residues identified by N-terminal sequencing of the cleaved His- Caspase-dependent cleavage of periplakin after induction of tagged fragment are indicated in bold and the caspase recognition site apoptosis. Madin–Darby canine kidney cells expressing tagged pe- is boxed, and the cleavage site is indicated by an arrowhead. The riplakin were grown to 80% confluency, and 1 mM staurosporine (STS) heptad repeat regions in periplakin are underlined. was applied for 1, 2, or 3 h. A caspase inhibitor zVAD-fmk (40 mM) was added to the culture medium 30 min prior to incubation with STS. The cleavage of periplakin was detectable at 2- and 3-h time points after STS addition (panels a, b, c: arrows in a and b), but the preincubation although unconventional for caspase 6, is located in the with caspase inhibitor prevented the appearance of STS-mediated middle of a potential loop that connects the last two heptad cleavage products. Actin (d) is shown as an indicator of cell count, repeats of the periplakin rod domain (Fig 8b). which significantly increased in the culture medium after induction of The caspase 6-generated C-terminal fragment was pu- apoptosis. rified (Fig 8a, line 3) and used for IF binding experiments along with other periplakin constructs (detailed in Fig 2). It exhibited binding properties similar to periplakin tail (Pt) alone (data not shown).

Discussion

In this study, we demonstrate the direct molecular interac- tion between actin and periplakin. We also show that oligomerization of periplakin is required for its efficient direct binding to both vimentin and keratin 8, as well as to vimentin Figure 7 Periplakin is specifically cleaved by caspase 6 in vitro. Madin–Dar- and keratin 8/18 IF. Moreover, caspase 6-mediated cleav- by canine kidney cell lysate containing tagged periplakin was subjected age of the periplakin at the end of its rod domain is ex- to digestion with recombinant caspases 1–10 (lanes 1–10, respective- pected to release the remaining N-terminal part of the ly). Lane 0 represents cell lysate incubated without caspase enzyme. molecule from IF while retaining the ability of the cleaved tail The caspase digests were separated by sodium dodecyl sulfate-po- lyacrylamide gel electrophoresis and immunoblotted with mAb PPL- to interact with keratin 14. 488 (a) and anti-HA-tag antibody (b). The antibody PPL-488 revealed a It should be noted that we found differences in the ability 180-kDa cleavage product, and the HA-tag antibody demonstrated the of periplakin to bind individual IF proteins in the overlay appearance of a short C-terminal cleavage product of 15–20 kDa in the assay versus assembled IF in the co-sedimentation assay. caspase 6-treated sample. In particular, periplakin was able to interact with simple IF proteins (vimentin and keratin 8) in both their forms, where- region contains eight aspartate residues, potentially serving as interaction with keratin 14, which is expressed as caspase cleavage sites. Therefore, a recombinant pe- in stratified epithelia, was detected only in the case of riplakin fragment, containing the last third of the rod and the the monomeric protein. On the contrary, desmoplakin was tail domain (Pr231t, see Fig 1), was used to determine the shown to interact with polymerized IF proteins but not with caspase 6 cleavage site. their monomers (Fontao et al, 2003), involving recognition Caspase 6 cleaved the recombinant protein into two sites located primarily in the rod domain of keratins. The fragments (Fig 8a, lane 2), the smaller of which showed authors speculated that the interaction was affected by the electrophoretic mobility identical to the C-terminal fragment tertiary structure induced by heterodimerization of IF pro- cleaved off the full-length recombinant periplakin by the teins. By contrast, recognition site(s) for the periplakin on caspase 6. After transfer to polyvinylidene fluoride (PVDF) keratin 14 might be located in the keratin head or tail do- membrane, the corresponding band was cut out and sub- main. Also, it has been shown that desmosomal proteins jected to N-terminal amino acid sequencing. The sequence associate predominantly with type II epidermal keratins SGTNHD matched with periplakin amino acid residues from (Kouklis et al, 1994; Meng et al, 1997), whereas hem- 1604 to 1609, and is preceded by an aspartic acid residue in idesmosomal proteins interact with type I keratins (Aho position 1603. The recognition amino acid sequence, TVAD, and Uitto, 1999; Geerts et al, 1999). Our data show that 52 KALININ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY periplakin can interact with both keratin 8 (type II) and that preferred by caspases 1 and 2, in vitro periplakin was keratin 14 (type I). not cleaved by these caspases. Although several studies have revealed multiple interac- It is known that after induction of apoptosis, cytoskeletal tions for the periplakin C-terminal region, the molecular in- elements and cell adhesion structures become degraded teractions of the N-terminal domain have been studied less through the action of caspases. Plakin proteins plectin, extensively. Although the actin-binding calponin homology desmoplakin, and periplakin are all cleaved by caspases repeats are not present in the periplakin N-terminus, the Y- but, surprisingly, different caspases preferentially cleave domain of periplakin shows considerable homology to each of these proteins (Stegh et al, 2000; Weiske et al, 2001; spectrin repeats, potentially mediating the specific bind- Aho, 2004b). Whereas plectin and desmoplakin are cleaved ing of periplakin head domain to filamentous actin. Co- by multiple caspases, periplakin was cleaved only by ca- localization with cortical actin even after depolymeriza- spase 6 in vitro, which also cleaves the majority of IF pro- tion of F-actin suggests either the direct or indirect teins. Thus, when cytoskeletal structures are reorganized interaction between these molecules (DiColandrea et al, during development, growth, and tissue remodeling, peripl- 2000). Our results indicate direct binding of periplakin to akin degradation can take place together with IF proteins, actin filaments. whereas a different set of caspases is involved in the deg- Taking into consideration the ability of periplakin to bind radation of other plakin proteins. It is worth noting that both IF and microfilaments, it is worth noting that periplakin specific binding of PKB/c-Akt to periplakin (van den Heuvel seems to exhibit a preference for either one or the other et al, 2002) may protect periplakin from cleavage by ca- depending on cell type and/or the physiological state of the spases in some circumstances, as the PKB/c-Akt is known cells. For example, full-length periplakin shows character- for its antiapoptotic function (Brazil et al, 2002; Le Gall et al, istic IF staining in simple epithelial (COS7) cells (Di- 2003). Colandrea et al, 2000; Karashima and Watt, 2002), but it Finally, periplakin has been characterized as one of five has membrane localization in keratinocytes (Ma and Sun, plakin proteins targeted by autoantibodies in paraneoplastic 1986; Ruhrberg et al, 1997; DiColandrea et al, 2000), al- pemphigus (PNP) (Anhalt et al, 1990; Anhalt, 1997; Mahon- though overexpression of periplakin can lead to its redis- ey et al, 1998). The etiology of this autoimmune disease is tribution to IF in keratinocytes as well (DiColandrea et al, not known. Clustering of intracellular molecules in the sur- 2000). In our studies, in sparse cultures of kidney (MDCK) face blebs of apoptotic cells may expose the antigens and epithelial cells, overexpressed periplakin showed filament- their novel apoptotic fragments with non-tolerated struc- ous cytoplasmic distribution, suggestive of co-localization tures to the immune system (Casciola-Rosen et al, 1994). with IF, whereas in confluent cultures periplakin staining was Defective clearance or reduced anti-inflammatory conse- concentrated at the cell plasma membrane region. As quences of apoptotic material has been suggested to pro- shown in this work, differences in periplakin-binding ability vide a susceptibility factor to initiate a primary immune to keratin 8/18 and vimentin IF, compared with K5/14 IF, can response (Rosen and Casciola-Rosen, 1999). It remains to shed light on how periplakin subcellular localization is reg- be studied as to whether defects in the clearance of apo- ulated. Besides, caspase-mediated cleavage of periplakin, ptotic cells in general, and especially the defective elimina- disconnecting its IF-binding tail from the rest of the mole- tion of periplakin cleavage products, play a role in the onset cule, might mediate its redistribution from IF to the plasma of autoimmune response to plakin proteins in PNP. membrane in maturing cultures or in terminally differentiat- ing keratinocytes, as was shown for confluent cultures of MDCK cells in this work. Materials and Methods Among caspases 1–10 and 14, only caspase 6 cleaved Generation of cDNA constructs for bacterial expression and periplakin in vitro. Prevention of the cleavage by specific protein purification Full-length or fragments of human periplakin caspase 6 inhibitor in vivo strongly supports the role of ca- (see Fig 1) and human vimentin and keratins 8, 18, 5, 14, 1, and spase 6 in the cleavage of the periplakin, although involve- 10 were cloned into pET-23 bacterial expression vector (Novagen, ment of other caspases besides those tested here cannot Inc., Madison, Wisconsin) in-frame with C-terminal 6-His tag, ex- be ruled out. Caspase 6 cleaved human periplakin at an pressed in bacteria and purified as described (Kalinin et al, 2004). Proteins were resolved on 4%–12% Bis–Tris NuPAGE gels with unconventional sequence TVAD, which is located in the MOPS running buffer (Invitrogen, Carlsbad, California). middle of a potential loop, connecting the last two heptad repeat regions of the periplakin rod domain. Before deter- Dot-blot overlay assay IF proteins (1 mg each) were applied to mining the cleavage site, another site, LEID-1634, was PVDF membrane using the 96-well Bio-Dot Microfiltration System considered as a possible consensus sequence for caspase (BioRad Laboratories, Hercules, California). After blocking, the membrane was incubated with the purified periplakin fragments at 6. This more conventional sequence is, however, located in 5 mg per mL for 1 h at room temperature (RT). Bound periplakin a coiled-coil region of the periplakin rod domain, possibly was detected using chicken anti-periplakin tail antibodies and preventing the access of the caspase. Peptide substrates horseradish peroxidase (HRP)-conjugated anti-chicken secondary for caspases have been extensively tested (Talanian et al, antibodies (procedures were identical to the western protocol). The 1997). Ac-YVAD-pNA was preferred by caspase 1 and equal loading of IF proteins was verified by Amido Black staining of Ac-VDVAD-pNA by caspase 2, but these peptides were not the membrane. cleaved by caspase 6. In type I keratins, peptide sequences F-actin and IF co-sedimentation assays The Actin-Binding Pro- VEMD and VEVD, and in lamins VEID and VEVD, are tein Biochem Kit was purchased from (Denver, Colo- cleaved by caspase 6 (see Caulin et al, 1997). Although the rado) and F-actin was assembled according to the manufacturer’s caspase-recognition site in periplakin, TVAD, is closest to instructions. Typically, 1 mg of test protein was added to 5–10 mgof 124 : 1 JANUARY 2005 CASPASE CLEAVAGE OF PERIPLAKIN CYTOLINKER 53 freshly assembled F-actin in 5 mM Tris-HCl, pH 8.0, 50 mM KCl, Induction of apoptosis MDCK cells expressing tagged periplakin 1 mM MgCl2, 1 mM ATP, and 0.2 mM CaCl2. Samples were incu- were plated on the 60-mm tissue culture plates and grown to 80% bated at RT for 20 min and ultracentrifuged for 30 min at 29 p.s.i. confluency. Apoptosis was induced by 1 mM STS (Sigma) applied (about 175,000 g) in a Beckman Airfuge (Beckman Coulter, Full- from 1000 stock in dimethyl sulphoxide (DMSO). A cell-perme- erton, California). Supernatants (non-bound protein) and pellets able caspase inhibitor zVAD-fmk (Biomol) was applied to the cells (cytoskeleton-bound protein) were analyzed by SDS-PAGE. To de- in 40 mM concentration from the 500 stock 30 min prior to the termine the binding parameters, a range of test protein concen- STS addition. A control culture was treated with an equal volume of trations was used, and the amount of protein in the pellet and in the DMSO. supernatant was quantitated by densitometry of Coomassie Blue- stained gels. Data were analyzed using GraphPad Prism software IIF microscopy For the IIF, the cultured cells on the chamber (GraphPad Software, San Diego, California). slides were fixed with methanol for 5–10 min and, after two washes IF were assembled by step dialysis as described (Yamada et al, with PBS, were further permeabilized with 0.1% Triton X-100 in 2003), and binding experiments were carried out as described PBS for 5 min at RT, followed by three washes with PBS. Blocking above, but in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5 mM b- was carried out with 1% bovine serum albumin (BSA) in PBS for 1 h 1 mercaptoethanol (b-ME). at RT, followed by primary antibody incubation at 8 C overnight. Slides were washed three times with PBS, each time for 5 min, Cross-linking experiments Purified proteins at 100 mg per mL in followed by the secondary antibody incubation at RT for 1 h. After 25 mM Triethanolamine-HCl, pH 7.8 were cross-linked with 2 mM three washes with PBS, slides were mounted and studied under a Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (Sulfo-BSO- fluorescent microscope (Axioskop, Carl Zeiss, Inc., Thornwood, COES) (Pierce, Rockford, Illinois, USA) at room temperature for New York). The images were stored with ImagePro Plus 4.0 im- 30 min, followed by SDS-PAGE on 4%–12% Bis–Tris NuPAGE gels aging software (Media Cybernetics, Silver Spring, Maryland) and with MOPS running buffer (Invitrogen). Protein bands were visu- processed with Photoshop 5.0 (Adobe Systems Inc., San Jose, alized by Coomassie Blue staining. California) and Canvas 7 (Deneba Software, Miami, Forida).

Size-exclusion chromatography The apparent molecular mass Cell extraction and western analysis Adherent cells on the 60- of recombinant proteins was estimated on Superdex 200 10 per mm tissue culture plates were briefly washed and extracted with 300 column (Amersham Biosciences, Piscataway, New Jersey) 150 mL of radioimmunoprecipitation-buffer: 50 mM Tris-HCl, pH calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), catalase 7.5; 150 mM NaCl; 1% Nonidet P-40 (NP-40); 0.5% deoxycholate; (232 kDa), lactate dehydrogenase (140 kDa), bovine serum albumin 0.1% SDS; protease inhibitor Complete (Roche Diagnostics), one (66 kDa) (all from Amersham Biosciences), and chymotrypsinogen tablet dissolved into 10 mL of buffer. After extraction and sonicat- (25 kDa) (Sigma, St Louis, MO, USA). The retention factor (KD) was ion, cell lysate was separated from the insoluble pellet through calculated as KD ¼ (VeVo)/(VtVo), where Ve is the protein elution centrifugation for 15 min at 14,000 g at 81C. The soluble fraction volume, Vo is the column void volume, and Vt is the total column was mixed with 2 SDS-Laemmli sample buffer (Bio-Rad) sup- bed volume. plemented with b-ME (sample buffer (SB)). Floating cells from the culture medium were collected through a brief centrifugation and Expression of tagged periplakin in MDCK cells The full-length dissolved into 150 mL of SB. All samples were heated for 15 min at periplakin cDNA in pcDNA3.1 (Aho, 2004a) was tagged with the N- 961C before loading on the PAGE. terminal FLAG-epitope by inserting a synthetic double-stranded For western analysis, 10-mL aliquots of cell lysates were sep- oligonucleotide with the EcoRI-compatible ends into the partially arated either on SDS–5% PAGE or on the 4%–20% gradient PAGE EcoRI-digested periplakin-pcDNA3.1 plasmid. The FLAG-peripl- and transferred onto PVDF membranes (PerkinElmer Life Scienc- akin cDNA was released with HindIII-NotI digestion and ligated into es, Boston, Massachussets). The membranes were blocked for 1 h the HindIII-NotI digested pCEP4t. A C-terminal HA-tag was insert- at RT in PBS, 1% BSA, and 5% non-fat dried milk. After the pri- ed by ligation of a double-stranded synthetic oligonucleotide with mary antibody incubation in 1% BSA in PBS overnight at 81C, the NotI-compatible ends into the NotI-digested FLAG-periplakin- membranes were washed four times, 10 min each wash, in 0.5% pCEP4t. The pCEP4 (Invitrogen) had been modified to drive the Tween 20 in Tris-buffered saline, followed by incubations for 1 h at expression of the transgene from a minimal CMV promoter under RTwith the secondary antibody, and then washed again. The signal the control of seven repeats of tet response element, resulting in was developed using Western Lightning Chemiluminescence Re- the expression vector pCEP4t. The vector contains the EBNA-1 agent Plus (PerkinElmer Life Sciences). for episomal autonomous replication in mammalian cells and a hygromycin gene for the selection of transfected cells. Compe- In vitro cleavage of periplakin by recombinant caspases Re- tent Escherichia coli cells, JM109 (Promega, Madison, Wisconsin) combinant human caspases 1–10 were purchased from Biomol. To or XL-1 Blue (Stratagene, La Jolla, California), were used for trans- examine periplakin as a substrate for caspases, 10 mg of MDCK formation, and plasmids were purified using the Wizard Miniprep cell lysate was diluted into 40 mL of the caspase assay buffer (50 Kit (Promega). Purified plasmids were sequenced to confirm the in- mM HEPES, pH 7.4; 100 mM NaCl; 10 mM dithiothreitol; 1 mM frame ligation of the inserts. EDTA; 0.1% CHAPS; 10% glycerol) containing 2 U per mL of the The Tet-Off MDCK cell line was purchased from Clontech (San recombinant caspases 1–10. After incubation for 18 h at 301C, the Diego, California) and grown according to the provider’s instruc- reaction was stopped by boiling in reducing Laemmli sample buffer tions. For the transfections, cells were plated on the 60-mm plates, (Bio-Rad), and the proteins were separated through SDS-PAGE. and the FuGENE 6 Transfection Reagent (Roche Diagnostics, In- For caspase 6 cleavage site determination, 0.5 mg of recom- dianapolis, Idiana) was used in the ratio of 1 mg of DNA to 3 mLof binant protein Pr231t was incubated with 1500 U of the recom- transfection reagent, following the manufacturer’s instructions. binant caspase 6 for 18 h at 301C in 1 mL of the caspase buffer. A Doxycycline was included in the transfection medium, and 24 h 10-mL aliquot was resolved on SDS-gel and transferred to se- after transfection, selection was started with hygromycin. Cultures quence-grade PVDF membrane by western blotting. Proteins on were trypsinized and replated before reaching confluency, usually the membrane were stained with 0.1% Amido Black (Sigma) in every 3–4 d. After three to five passages, no adherent cells re- 25% ethanol, 10% acetic acid for 1 min, and background was mained in the untransfected control cultures. For the induction of removed by two 10-min washes in 50% ethanol, 2% acetic acid. the transgene expression, cells were trypsinized and plated with- The membrane was air-dried, the band corresponding to the C- out doxycycline, allowed to grow to 70% confluency, trypsinized terminal fragment was cut out, and its N-terminal amino acid se- again, and replated on 60-mm tissue culture plates before har- quence was determined on a Beckman 6300 analyzer (Beckman vesting for the western blotting, and replated into the 4-well cham- Coulter). The C-terminal fragment of periplakin was purified from ber slides for indirect IIF. the remainder of caspase 6-cleaved sample on Ni–NTA beads 54 KALININ ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

(Qiagen, Valencia, California) according to the manufacturer’s in- Beekman J, Bakema J, van der Linden J, et al: Modulation of FcgRI (CD64) ligand structions and used for the overlay and co-sedimentation assays. binding by blocking peptides of periplakin. J Biol Chem 279:33875– 33881, 2004a Beekman J, Bakema J, van de Winkel JG, Leusen JH, Direct interaction be- Primary and secondary antibodies The following commercially tween FcgRI (CD64) and periplakin controls receptor endocytosis and available antibodies were used: mouse monoclonal anti-FLAG M2 ligand binding capacity. Proc Natl Acad Sci USA 101:10392–10397, (1:3000; Stratagene), rabbit HA-tag (1:3000; Covance, Richmond, 2004b California), mouse monoclonal HA-tag (1;10,000; Covance), and Beil M, Leser J, Lutz MP, et al: Caspase 8-mediated cleavage of plectin precedes mouse monoclonal anti-actin antibody clone C4 (0.2 pg per mL; F-actin breakdown in acinar cells during pancreatitis. Am J Physiol Gas- Boehringer/Roche Diagnostics). trointest Liver Physiol 282:G450–G460, 2002 Bornslaeger EA, Corcoran CM, Stappenbeck TS, Green KJ: Breaking the con- The polyclonal chicken antibody against periplakin C-terminus nection: Displacement of the desmosomal plaque protein desmoplakin was produced by Aves Labs, Inc. (Tigard, Oregon). Purified re- from cell–cell interfaces disrupts anchorage of intermediate filament bun- combinant periplakin tail domain (periplakin aa 1638–1756, without dles and alters intercellular junction assembly. J Cell Biol 134:985–1001, the His tag) was used for immunization. The antibody was affinity 1996 purified and used at 1 mg per mL. The GST–periplakin fusion pro- Brazil DP, Park J, Hemmings BA: PKB binding proteins. Getting in on the Akt. Cell tein (periplakin aa 1548–1756) was used to immunize mice, and a 111:293–303, 2002 hybridoma cell line-producing monoclonal antibody PPL-Y88 rec- Byun Y, Chen F, Chang R, Trivedi M, Green KJ, Cryns VL: Caspase cleavage ognizing periplakin aa 1548–1583 was selected (Aho, 2004a). of vimentin disrupts intermediate filaments and promotes apoptosis. Cell HRP-conjugated goat anti-chicken IgY was purchased from Death Differ 8:443–450, 2001 Aves Labs, Inc., and used at 1:25,000 dilution. HRP-conjugated Casciola-Rosen LA, Anhalt G, Rosen A: Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on secondary antibodies against mouse, rabbit, and goat IgG were apoptotic keratinocytes. J Exp Med 179, 1994 purchased from Jackson ImmunoResearch Laboratories (West Caulin C, Salveson GS, Oshima RG: Caspase cleavage of keratin 18 and reor- Grove, Pennsylvania), and used for the western blotting in 1:25,000 ganization of intermediate filaments during epithelial cell apoptosis. J Cell dilution. Species-specific secondary antibodies, conjugated with Biol 138:1379–1394, 1997 Texas Red and fluorescein isothiocyanate, were also purchased Chen F, Chang R, Trivedi M, Capetanaki Y, Cryns VL: Caspase proteolysis of from Jackson ImmunoResearch Laboratories, and used for IIF in desmin produces a dominant-negative inhibitor of intermediate filaments 1:500 dilution in PBS–1% BSA. Nuclei were visualized with DAPI, and promotes apoptosis. J Biol Chem 278:6848–6853, 2003 which was included in the secondary antibody incubation. Choi H-J, Park-Snyder S, Pascoe LT, Green KJ, Weis WI: Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure. Nat Struct Biol 9:612–620, 2002 DiColandrea T, Karashima T, Ma¨ a¨ tta¨ A, Watt FM: Subcellular distribution of This work is dedicated to the memory of Dr Peter M. Steinert, who envoplakin and periplakin: Insights into their role as precursors of the initially established the collaboration leading to the results presented in epidermal cornified envelope. J Cell Biol 151:573–585, 2000 this paper. We thank Dr Lyuben N. Marekov for amino acid sequencing. Feng GJ, Kellett E, Scorer CA, Wilde J, White JH, Milligan G: Selective interac- This work was supported by RO1 33588 from NIH, NIAMS (S. A.). tions between helix VIII of the human mu opioid receptors and the C- terminus of periplakin disrupt G protein activation. 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