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Originally published In Press as doi:10.1074/jbc.M303896200 on July 9, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36707-36717, September 19, 2003
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Characterization of Periphilin, a Widespread, Highly Insoluble Nuclear Protein and Potential Constituent of the Keratinocyte Cornified Envelope*

Shideh Kazerounian and Sirpa Aho {ddagger}

From the Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, April 14, 2003 , and in revised form, July 1, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
While keratinocytes go through the terminal differentiation and move toward the outer layers of epidermis, multiple proteins become sequentially incorporated into the cornified cell envelope. We have identified through yeast two-hybrid screening a novel protein, periphilin, interacting with periplakin, which is known as a precursor of the cornified cell envelope. Periphilin gene at chromosome 12q12 gives rise to multiple alternatively spliced transcripts. A monoclonal antibody detected the keratinocyte-specific periphilin isoform in undifferentiated keratinocytes in speckle-type nuclear granules and at the nuclear membrane, but in differentiated keratinocytes periphilin localized to the cell periphery and at cell-cell junctions, colocalizing there with periplakin. From cultured keratinocytes, periphilin was solubilized only after urea extraction, indicating the highly insoluble character of this protein. The nuclear localization, mediated through the N-terminal sequences of periphilin protein, is a prerequisite for the formation of insoluble complexes. Although the globular N terminus of periphilin was necessary for the interaction with the periplakin tail, the keratinocyte-specific C terminus was responsible for the homodimerization. The C-terminal helical domain, composed of multiple heptad repeats, serves as a substrate for cross-linking by transglutaminases but also was specifically cleaved by caspase-5 in vitro. In conclusion, the localization pattern and insolubility of periphilin indicate that this novel protein is potentially involved in epithelial differentiation and contributes to epidermal integrity and barrier formation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cornified cell envelope consists of proteins such as periplakin, envoplakin, involucrin, loricrin, and small proline-rich proteins, which are gradually deposited under the inner side of the plasma membrane as keratinocytes go through terminal differentiation and move to the higher epidermal layers. These proteins are cross-linked in a Ca2+-dependent reaction that is catalyzed by the epidermal transglutaminases to provide a protective layer between the environment and the living layers of the skin. The water barrier function of this envelope is maintained by a thick layer of lipids attached to the exterior of the cornified envelope (1, 2). Envoplakin and periplakin act as an interdesmosomal scaffold on which the cornified envelope is assembled (2, 3). Envoplakin and periplakin also form covalent bonds with ceramide lipids at the plasma membrane, which further supports their role in the envelope assembly process (4).

Several recently characterized molecular interactions indicate that periplakin may serve as a docking platform for a variety of unrelated proteins. An interaction with protein kinase B (PKB/c-Akt)1 has been reported, and it was suggested that periplakin can function in relocating this kinase into various cell compartments (5). PKB signaling plays a crucial role in providing cellular protection against apoptosis. PKB becomes activated at the plasma membrane by phosphoinositide 3-kinase-mediated production of phosphoinositol lipids. Following activation, PKB needs to translocate to other cellular compartments. In addition to acting as a docking site, periplakin may also function as a shuttle for the delivery of PKB to various subcellular compartments (5). Interaction with the 300-kDa mannose 6-phosphate receptor (M6PR300) (6) also indicates a role in transport and docking. MPRs are known to function in the targeting of newly synthesized lysosomal enzymes through their ability to cycle between the trans-Golgi network, where the ligands are bound, and endosomes, where the ligands are released (7). In addition, MPRs cycle between cell surface and endosomes. MPRs are also involved in the constitutive secretory pathway of small transport vesicles from the trans-Golgi network to the plasma membrane. Thus it is suggested that periplakin facilitates the transport of vesicles or that it functions as a scaffold at the plasma membrane during terminal differentiation of epidermal keratinocytes, either during the degradation of cellular compartments or the assembly of the cornified envelope. The intermediate filaments, composed of keratins and vimentin, form the major scaffold within both epithelial and mesenchymal cells. The colocalization of periplakin with the intermediate filaments (8, 9) and the direct molecular interaction with keratin 8 and vimentin (5, 10) emphasize a role for periplakin as an intermediate filament binding protein similar to plectin (11). The central rod domain of periplakin, predicted to form a coiled-coil structure, is responsible for homodimerization, but periplakin is also able to heterodimerize with envoplakin (8, 12). Envoplakin is one of the early precursors of the cornified cell envelope. Although, envoplakin alone is not able to localize to the plasma membrane or bind to the intermediate filaments, it has been suggested that a dependence on heterodimerization with periplakin can facilitate such actions (8).

To identify proteins interacting with periplakin, we executed a yeast two-hybrid screening with the periplakin C-terminal domain (10). Here we report the characterization of a novel protein that colocalizes with periplakin at the cell-cell junctions in the outer granular cell layers of epidermis but is also a prominent nuclear protein. The novel protein, periphilin, may have a periplakin-independent functional role in the cell nucleus, but although the terminally differentiated keratinocytes become flattened within the upper granular layer of epidermis and the nuclei become disintegrated, the molecular complexes containing this highly insoluble protein may bind to periplakin at the plasma membrane and become incorporated into the cornified cell envelope.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Two-hybrid Assays—For the two-hybrid bait construct, a cDNA clone encoding periplakin C-terminal region, aa 1584–1756, was inserted into pGB-MEL1 (10). Human foreskin keratinocyte cDNA library in the GAL4-AD vector was purchased from Clontech and amplified according to the manufacturer's instructions.

Yeast two-hybrid assays were performed according to the Clontech yeast two-hybrid manual, using the Frozen-EZ Yeast Transformation II kit (Zymo Research) for preparing competent yeast cells. Saccharomyces cerevisiae YRG-2 cells (Stratagene) were cotransformed with 1 µg of GAL4 binding domain and GAL4 activation domain fusion constructs. Cells were plated on double-minus plates (–Trp, –Leu) and triple-minus plates (–Trp, –Leu, –His), and colonies were allowed to grow at 30 °C for 3–5 days. The positive interaction between two proteins was detected by the appearance of large pink colonies on triple-minus plates. The ingredients for yeast media were purchased from Difco and from Bio 101, Inc. (Vista, CA).

Periphilin Expression Constructs—Plasmid DNA of the cDNA library was used as a template for polymerase chain reaction (PCR) with DyNAzyme EXT DNA polymerase (Finnzymes OY, MJ Research, Inc.), and PCR was executed according to the manufacturer's instructions. To generate periphilin deletion constructs, forward primers 5'-GAA TTC GAT GAA TCT GGT TAT AGA TGG (exon 5) and 5'-GAA TTC GAT AAA GAG AGG CCT GTC CAG (exon 8) and reverse primers 5'-GTC GAC TAT TTC ATG TAA ATT CTG C (exon 11 at the cryptic splicing site) and 5'-GTC GAC TAA AAA GGC TCT CCA AAA TCT (exon 11 at the translation termination site) were used to amplify periphilin domains encoding aa 87–374, 197–374, and 197–349. The EcoRI and SalI sites were incorporated into primers and are underlined above. The PCR fragments were purified and subcloned into pBluescript II KS (Stratagene). The EcoRI-SalI fragments from pBluescript were purified and cloned in-frame into pGEX-4T-1 (Amersham Biosciences), pGB-MEL1 (13), and pGAD424 (Clontech) vectors. A BamHI fragment (periphilin aa 265–374) from the pGAD10 periphilin two-hybrid clone (aa 8–374) was separated and subcloned into the GAL4-AD vector and into the pGEX-5X-2 vector.

C-terminally Myc-tagged periphilin expression construct was generated by PCR using an exon 1-specific primer, 5'-CTC CCG GAG GGC CAG AGT, and a reverse primer complementary to the end of the coding sequence in exon 11, 5'-AGT CGA CTA ATT CAA GTC CTC TTC AGA AAT GAG CTT TTG CTC AAA AGG CTC TCC AAA ATC TTG. The sequence encoding the Myc-tag is underlined. The PCR product was first ligated into EcoRV-cut and T-tailed pcDNA3 vector, and then the HindIII-NotI insert was isolated and ligated to the HindIII-NotI-digested pCEP4 (Invitrogen), which had been modified to drive the expression of the transgene from a minimal cytomegalovirus promoter under the control of seven repeats of the tet response element. The pCEP4 vector contains the EBNA-1 gene for autonomous replication in mammalian cells and a hygromycin gene for the selection of transfected cells. For N-terminally green fluorescent protein (GFP)-tagged periphilin constructs, a fragment of NheI-HindIII digest was separated from pEGFP-C2 (Clontech) and inserted between the NheI and HindIII sites in front of the periphilin cDNA in pCEP4-tet vector.

Competent JM109 (Promega) or XL-1 Blue (Stratagene) Escherichia coli cells were used for transformations, and plasmids were purified using Wizard Miniprep Kit (Promega). Purified plasmids were sequenced to confirm the in-frame ligation of the inserts.

Cell Cultures and Transfections—Human foreskin keratinocytes were isolated by the cell culture core unit in the Department of Dermatology at Thomas Jefferson University. Keratinocytes at passage 1 were used for the experiments described here. After separating from the epidermis with trypsin treatment, keratinocytes were plated on Matrigel-coated 8-well chamber slides and allowed to adhere and reach 50% confluency. The calcium-free keratinocyte growth medium (KGM) was supplemented with 60 µM, 150 µM, or 1 mM CaCl2 and applied on the keratinocytes for 2 days before preparing the slides for the indirect immunofluorescence (IIF) or harvesting the cells for protein extractions.

The Tet-Off cell lines were purchased from Clontech and grown according to the provider's instructions. For the transfections, cells were plated on the 60-mm plates, and FuGENETM 6 Transfection Reagent (Roche Diagnostics) was used in the ratio of 1 µg of DNA to 3 µl of transfection reagent, following the manufacturer's instructions. Doxycycline was included in the transfection medium, and 24 h after transfection, selection was started with hygromycin. Cultures were trypsinized and replated before reaching confluency, usually every 3–4 days. After 3–5 passages, no adherent cells were left in the untransfected control culture. For the induction of the transgene expression, cells were trypsinized and plated without doxycycline, left to grow to 70% confluency, trypsinized again, and replated on 60-mm tissue culture plates before harvesting for the Western blotting and into the 4-well chamber slides for IIF.

Primary and Secondary Antibodies Used for the Immunoassays—A glutathione S-transferase (GST)-periphilin (aa 265–374) fusion protein was expressed in XL-1 Blue (Stratagene) E. coli strain. The fusion protein was purified through binding to glutathione-Sepharose 4B (Amersham Biosciences or Cytoskeleton) according to manufacturer's instructions. The protein was eluted with glutathione elution buffer (Sigma), and the protein concentration was measured using Bio-Rad protein assay reagent.

Periphilin monoclonal antibody was obtained through immunizing mice with the GST-periphilin fusion protein (periphilin aa 265–374). The immunized mice were sacrificed for the hybridoma production (Hybridoma Core Facility, Wistar Institute, Philadelphia, PA). Four independent hybridomas were tested for their specificity; one hybridoma was cloned through the limited dilution, and a selected clone was grown in the large-scale culture. The culture supernatant was tested through enzyme-linked immunosorbent assay and used for Western blotting (1:10 dilution) and immunofluorescence assays (1:3 dilution).

Polyclonal antiserum against periplakin head domain was obtained by immunizing a rabbit (#3196, Alpha Diagnostic International, San Antonio, TX), with the peptide GRSSHVSKRARLQSPATKVK (periplakin aa 817–836), conjugated to keyhole limpet hemocyanin. The enzyme-linked immunosorbent assay titer (1:100,000) against the peptide was assayed. The antiserum was affinity-purified through binding to a Protein G affinity column (MabTrapTM kit, Amersham Biosciences). The polyclonal mouse serum against the C-terminal domain of periplakin was obtained through immunizing mice with a GST-periplakin fusion protein (periplakin aa 1548–1756), and a hybridoma cell line producing monoclonal antibody recognizing periplakin aa 1548–1583 was selected.2

For the Western blotting, GFP monoclonal antibody (Clontech) was used in 1:5,000 dilution, mouse monoclonal GST (NeoMarkers) was used in 1:10,000 dilution, and 0.2 pg/ml mouse monoclonal anti-actin antibody clone C4 (Roche Applied Science) was used. Secondary antibodies were purchased from Jackson Laboratories. Horseradish peroxidase-conjugated anti-mouse antibody was used for the Western blotting in 1:25,000 dilution. Species-specific secondary antibodies, conjugated with Texas Red and fluorescein isothiocyanate and used for IIF in 1:500 dilution in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA). Nuclei were visualized with DAPI (diamidinophenyl indole), which was included in the secondary antibody incubation.

Immunofluorescence Microscopy—For the IIF, a block of human foreskin was embedded in the OCT compound, frozen, and cut into 7-µm sections. Slides were either stored frozen at –20 °C, or used immediately. Tissue sections as well as the cultured cells on the chamber slides were fixed with methanol for 5–10 min and, after two washes with PBS, were further permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature (RT), followed by three washes with PBS. Blocking was done with 1% BSA in PBS for 1 h at RT, followed by the primary antibody incubation at 8 °C overnight. Slides were washed three times with PBS, each time for 5 min, followed by the secondary antibody incubation at RT for 1 h. After three washes with PBS, slides were mounted with Anti-Fade (Molecular Probes) and studied under a fluorescence microscope (Axioskop, Carl Zeiss, Inc). The images were stored with ImagePro Plus 4.0 imaging software (Media Cybernetics) and processed with Photoshop 5.0 (Adobe Systems Inc.) and Canvas 5 (Deneba Software).

Solubilization of Periphilin and Western Blot Analysis—To perform a serial extraction of periphilin from keratinocyte cultures, cells on 100-mm culture dishes were first briefly washed on ice with ice-cold PBS. Cells were extracted with 600 µl of the high salt buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1.5 M KCl; 1% Nonidet P-40; 0.5% sodium deoxycholate; 1% Triton X-100; protease inhibitor Complete (Roche Applied Science) 1 tablet dissolved into 10 ml of buffer) by collecting the cell layer with the cell scraper and after transferring to microcentrifuge tubes, cells were mixed by vortexing, then sonicated and incubated on ice for 30 min. The pellet was separated through microcentrifugation in a cold cabinet for 30 min at 14,000 rpm, and the supernatant was carefully collected. The pellet was resuspended in 600 µl of the urea buffer (50 mM Tris-HCl, pH 7.4; 8 M urea; 1% Triton X-100) and incubated on ice for 30 min before repeating the centrifugation step. The supernatant was carefully separated. The pellet was resuspended in SDS-Laemmli sample buffer (Bio-Rad) supplemented with {beta}-ME. An aliquot of each supernatant was mixed with the sample buffer, and the samples were heated for 10 min at 96 °C before separation through the SDS-PAGE.

For one-step extraction, cells were extracted with a radioimmunoprecipitation (RIPA)-buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.5% deoxycholate; 0.1% SDS). After extraction as described above, the soluble fraction was separated by centrifugation, and the pellet was further dissolved by resuspending into SDS-sample buffer and heated as described above.

For Western blotting, 10-µl aliquots of cell lysates were separated on SDS-10% PAGE and transferred onto polyvinylidene fluoride membranes. The polyvinylidene fluoride membranes were blocked for 1 h at RT in PBS, 1% BSA, 5% nonfat dry milk powder. After the primary antibody incubation in PBS 1% BSA overnight at 8 °C, the membranes were washed four times, 10 min each wash, in Tris-buffered saline/0.5% Tween 20, incubated for 1 h at RT with the secondary antibody, then washed again, and the signal was developed using Renaissance Western blot chemiluminescence reagent (PerkinElmer Life Sciences).

Analysis of Periphilin Transcripts—Human Multiple Tissue cDNA panels I and II were obtained from Clontech and used as templates for PCR analysis. A 10-fold serial dilution of a human keratinocyte cDNA library DNA was also used as a PCR template. Periphilin primers (p724) 5'-GGG ACG ATA TGA ATA TGA AAG and (p725) 5'-GAA ACT GCT GAA CCA CTT GA, specific for exons 3 and 8, respectively, and (p767) 5'-GAT AAA GAG AGG CCT GTC CAG and (p768) 5'-TAA AAA GGC TCT CCA AAA TCT, specific for exons 8 and 11, were used. PCR conditions were: 2 min at 95 °C, followed by 38 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. PCR was conducted using TaqDNA polymerase and the Q-solution provided with the kit (Qiagen). Ubinuclein-specific primers 5'-AGA AGC CAT GCA GTG ACA C and 5'-AGC TCT GGG TAG AAG AAC, producing a PCR fragment of 243 bp, were used as a control (14). The PCR products were separated on 1.5% agarose-TBE (Tris borate-EDTA) gels.

The Cross-linking Assay—To examine periphilin as transglutaminase (TGase) substrate, 200 ng of each GST fusion protein was incubated with 1.0 µg of TGase 2 (Sigma) in buffer containing 50 mM Tris-HCl, pH 7.4; 20 mM CaCl2; and 5 mM dithiothreitol for 45 min at 37 °C. The reaction was stopped by addition of Laemmli sample buffer supplemented with {beta}-ME (Bio-Rad) and heating at 96 °C for 10 min. Human foreskin keratinocytes, cultured in KGM (Clonetics) supplemented with 150 µM Ca2+, were briefly washed and extracted for 20 min on ice with RIPA buffer without protease inhibitors, sonicated on ice, and the lysate was cleared with microcentrifugation in a cold cabinet for 30 min at 14,000 rpm. An aliquot of the lysate (10 µg of protein) was used in the transglutaminase assay as described above.

In Vitro Cleavage of Periphilin by Recombinant Caspases—Recombinant human caspases 1–10 were purchased from Biomol (Plymouth Meeting, PA). To examine periphilin as a substrate for caspases, 15 ng of GST-periphilin (aa 197–374) fusion protein was diluted into 20 µl of the caspase assay buffer (50 mM HEPES, pH 7.4; 100 mM NaCl; 10 mM dithiothreitol; 1 mM EDTA; 0.1% CHAPS; 10% glycerol) containing 2 units/µl of the recombinant caspases 1–10. After incubation for 18 h at 30 °C, the reaction was stopped by boiling in reducing Laemmli sample buffer (Bio-Rad), and the proteins were separated through 4–20% SDS-PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of Periphilin cDNA—To further explore the functional role of periplakin in epidermal keratinocytes, we performed a yeast two-hybrid screening using a C-terminal region of periplakin as a bait. The periplakin extended linker domain, aa 1584–1756, identified keratin 8 and vimentin as interacting proteins (10). In addition, a novel cDNA clone encoding a previously unidentified polypeptide was isolated. After further characterization, we proposed to name this novel protein "periphilin," the name of which together with the symbol PPHLN1 for the human periphilin gene was approved October 25, 2002, by the international Nomenclature Committee of HUGO (the Human Genome Organization). Periphilin cDNA sequence and the deduced polypeptide were deposited into the GenBankTM data base (accession number AY157850 [GenBank] ).

Periphilin gene, currently included in Homo sapiens genomic contig NT009781, was initially identified through a Blast search in two overlapping genomic clones of chromosome 12q12, locating close but not within the type II keratin gene cluster in 12q13 (Fig. 1). The full-length periphilin cDNA consists of 14 exons, spanning 120 kb of genomic sequences. Transcripts generated through the alternative splicing of 5'-exons 2, 4, and 7 and 3'-exons 11–14, give rise to multiple protein products. The periphilin cDNA clone isolated through the yeast two-hybrid screening was lacking exons 2, 4, and 12–14 (Figs. 1 and 2). Of sequences present in the human EST (expressed sequence tags) data base, exon 2 is in 23%, exon 4 is in 49%, and exon 7 is in 75% of cDNAs covering the corresponding region. The exclusion of exon 2 deletes the first in-frame translation initiation codon. The second in-frame ATG encodes Met-8. Both ATG codons are preceded by an in-frame translation stop codon TAA. Sequences surrounding the two ATG codons (Fig. 2) display homology to, but do not completely match with, the optimum Kozak consensus sequence GCC(A/G)CCATGG (15).



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  		FIG. 1.
Periphilin genomic locus on chromosome 12q12. This locus, PPHLN1, gives rise to multiple transcripts. Periphilin transcripts with alternatively spliced exons 2, 4, 7, and 11–14, are abundant in the GenBankTM EST data base. The positions of primers used in Fig. 4 for the PCR analysis of the tissue-specific expression of the alternatively spliced periphilin transcripts are indicated below the two-hybrid cDNA.

 


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  		FIG. 2.
Periphilin cDNA and deduced amino acid structure. Translation can be initiated either from M1 or M8, depending on the presence or absence of exon 2. The alternatively spliced exons 2 and 4 are highlighted with strikethrough, and exon 7 is underlined. The positions of the introns are indicated by pointing arrowheads. The position of the cryptic splicing within exon 11 is shown by a horizontal arrowhead. The in-frame translation termination codons preceding the open reading frame and the poly-A addition signals are boxed. A putative nuclear localization signal (aa 110–116) and a potential nuclear export signal (aa 320–327) are highlighted in italics. Within the C-terminal half of periphilin four predicted helixes, composed of heptad repeats, are highlighted by bold and underlined. A tetrapeptide LFTD-273, a possible caspase recognition sequence, is highlighted with shading. Nucleotides are numbered at the beginning of each line. Numbering of the amino acid residues, beginning from the first in-frame ATG codon is shown on the left.

 



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  		FIG. 4.
Expression of periphilin transcripts in human tissues. Human multiple tissue cDNA panels I and II and a serial dilution of human keratinocyte cDNA library DNA were used as templates for PCR amplification. A, a primer pair (p724-p725) specific for exon 3 to exon 8 produced multiple bands representing transcripts containing the alternatively spliced exons 4 and 7 (627 bp), exon 4 only (570 bp), or exon 7 only (462 bp) or lacking both exons 4 and 7 (405 bp). B, a pair of primers (p767-p768) producing a PCR product from exon 8 to exon 11 (547 bp) was designed to detect specifically the 3'-end of the transcript isolated through the two-hybrid interaction. C, as a positive control, a pair of primers producing a 220-bp fragment from the 5'-end of the ubinuclein, a transcript expressed ubiquitously tissue-wide, was used.

 

Structural Features of Periphilin—Secondary structure prediction of periphilin polypeptide revealed a hydrophilic flexible N-terminal half, whereas the C-terminal half is composed of heptad repeats potentially forming four {alpha}-helixes (Fig. 2). On the nucleotide level, 80% identity along the 690-bp region of periphilin cDNA was detected with a cDNA encoding a protein similar to serine-threonine kinase 6/aurora-A/IPL1-related kinase (GenBankTM number XM222537). The homology domain in cDNA encodes periphilin aa 140–334, showing 64.6% homology and 57.4% identity along this 195-aa region. The C-terminal half of periphilin revealed structural homology to the homodimerization domain of vitellogenin proteins. Such conserved structural motifs are utilized by mammalian proteins, involved in the lipid transport and storage, to form dimerization interfaces (16).

Molecular Interactions of Periphilin—To map the domain of periphilin responsible for periplakin interaction, a series of periphilin deletion constructs was tested in the yeast two-hybrid assay (Fig. 3). Because exon 4 was not present in the original two-hybrid clone, and the deletion construct starting from exon 5 did not show interaction, we conclude that N-terminal sequences from Met-8 to Ser-31 are indispensable for the interaction with the periplakin C-terminal domain. The C-terminal half of periphilin contains heptad repeats, which potentially form {alpha}-helical conformation (Fig. 2). Because this region showed a low degree of structural homology with the dimerization domain of vitellogenins, we used a yeast two-hybrid assay to test the possible homodimerization of the periphilin C terminus. Indeed, the C-terminal region of periphilin, aa 265–374, was found sufficient for the dimerization, but the deletion of the C-terminal sequences (aa 350–374) completely abolished the ability for self-interaction. Through a cryptic splicing within exon 11, abolishing aa residues 350–374, the last 25 aa residues encoded by exon 11 are replaced by 117 aa residues encoded by exons 12–14, increasing the pI of the corresponding protein from 6.59 to 9.15. This type of periphilin isoform has been identified as a gastric cancer-associated gene Ga50 (GenBankTM number AY039238 [GenBank] ) (17). Another C-terminally truncated isoform, due to the deletion of the entire exon 11, has been detected from skin with melanotic melanoma (GenBankTM BC039832 [GenBank] ). Although the consequences on protein function are not known, the inability to dimerize may change protein solubility properties.



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  		FIG. 3.
Molecular interactions of periphilin. Periplakin C-terminal domain, used as a two-hybrid bait (GAL4-binding domain fusion protein), interacts with full periphilin (expressed in yeast as a GAL4-activation domain fusion protein). A set of N-terminal deletions of periphilin do not interact with periplakin, indicating that the N terminus of periphilin, aa 8–86, is indispensable for periplakin-periphilin interaction. When tested against each other for self-interaction, the N-terminally deleted periphilin constructs were fully functional, but the deletion of 25 C-terminal amino acids, aa 350–374, completely abolished the two-hybrid interaction. Triple plus signs (+++) indicate a strong interaction; a minus sign (–) indicates no interaction. Each bait construct was tested with an empty vector so as not to induce auto-activation. Cells from each cotransformation were plated on both double-minus (–Trp, –Leu) and triple-minus (–Trp, –Leu, –His) selection plates to verify successful cotransformation and test protein-protein interaction, respectively.

 

Tissue-specific Expression of Periphilin Transcripts—Periphilin transcripts, similar to the two-hybrid clone isolated in this study, without exons 4 and 12–14, but with exons 2, 7, and 11, have been identified from CD34+ hematopoietic stem cells (HSPC232, GenBankTM number AF151066 [GenBank] ; HSPC206, GenBankTM number AF151040 [GenBank] ). To study the tissue-specific expression of the alternatively spliced periphilin transcripts, we designed two pairs of primers specifically amplifying either the 5'- or the 3'-half of periphilin cDNA (Fig. 1). Exon 3, containing the translation initiation site, and exon 8, present in all periphilin transcripts, were selected as targets for the 5'-end primers. Human Multiple Tissue cDNA panels as templates for PCR with the 5'-end primers (724–725) gave rise to four major bands from all tissues (Fig. 4A). The upper doublet corresponded to the transcript with exon 4, with (627 bp) or without (570 bp) exon 7, and the lower doublet represented the transcripts without exon 4 and with (462 bp) or without (405 bp) exon 7. The major band obtained from keratinocyte cDNA as a PCR template (462 bp) corresponded to the transcript with exon 7 but without exon 4. The 3'-end primers (767 and 768) produced a PCR product (547 bp) only when keratinocyte cDNA was used as a template (Fig. 4B). Because the primers 725 and 767 both are specific to exon 8, we conclude that the 3'-end of exon 11 is unique for the transcript present in keratinocytes.

Detection of Periphilin in Human Epidermis and Epidermal Keratinocytes—A GST-periphilin (aa 265–374) fusion protein was used to immunize mice. After the fusion, four hybridomas were obtained, one of which was chosen for cloning through the limited dilution. Culture supernatant from each hybridoma was tested on the Western blot against a set of GST-periphilin fusion proteins. Fig. 5A demonstrates that PHL mAb recognized the GST fusion proteins with periphilin aa 197–374 and 265–374 but did not recognize a C-terminally deleted fusion protein with periphilin aa 197–349. A degradation product of GST-periphilin aa 265–374, observed in the Coomassie staining and by the GST-specific antibody, was not detected with the monoclonal antibody specific for the extreme C terminus of periphilin. All four hybridoma supernatants gave identical recognition pattern on the Western blot (data not shown), indicating that, within the periphilin C terminus, the last 25 aa residues form the strongest antigenic epitope. Because the cryptic splicing within exon 11 takes place at aa 349, removing the last 25 aa residues encoded by this exon, the PHL mAb is specific for the keratinocyte-specific isoform of periphilin (Fig. 5B). The sequential extraction of primary human keratinocytes revealed that periphilin is not a soluble protein. On the Western blot the PHL mAb demonstrated a distinct band, which was solubilized from keratinocyte lysate with urea extraction (Fig. 5C). From the cells cultured in low Ca2+ medium, additional bands became soluble with boiling in the SDS-sample buffer. Also, periphilin protein was present equally in the cells grown in low and high Ca2+ conditions.



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  		FIG. 5.
Monoclonal antibody SK30 recognizes an epitope within the 25-aa C-terminal peptide of periphilin. A, GST fusion proteins with periphilin aa 197–374 (lane 1), aa 265–374 (lane 2), aa 197–349 (lane 3), and GST alone (lane 4), were probed with the GST-specific antibody (GST mAb) and with periphilin monoclonal antibody (PHL mAb), and visualized through Coomassie Blue staining. Loaded per lane were 40 ng of each protein for the Western blotting, and 200 ng of each protein for Coomassie Blue staining. B, schematic presentation of the GST fusion proteins, tested in A. PHL mAb specifically recognizes an epitope within the C-terminal aa 350–374. C, PHL mAb recognizes periphilin, extracted from cultured keratinocytes grown in low (60 µM) and high (1 mM) calcium conditions. Keratinocyte cultures were washed and serially extracted with equal volumes of high salt buffer (lane 1), urea buffer (lane 2), and SDS-sample buffer (lane 3). The hybridoma supernatant containing PHL mAb was used for Western blotting in 1:10 dilution.

 

The IIF analysis of frozen sections of neonatal foreskin revealed a dual subcellular localization for periphilin protein (Fig. 6, a–d). A prominent nuclear staining was evident throughout the epidermis as well as in nuclei of dermal fibroblasts. In addition, within the granular cell layer, periphilin antibody revealed a signal localizing to the cell periphery of the flattened keratinocytes. Mouse polyclonal antibody against the C-terminal domain of periplakin revealed a similar staining pattern but also highlighted cell-cell contacts in the spinous cell layer of epidermis (Fig. 6, e and f). Cross-sections of the excretory duct of sweat gland, traversing through the dermis, revealed the strongest signal at the apical side of the epithelial cells lining the ducts (Fig. 6, g and h). Again, periplakin antibody detected a similar staining pattern in a parallel tissue section (Fig. 6, i and j).



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  		FIG. 6.
Immunolocalization of periphilin in human skin. PHL mAb demonstrates strong nuclear staining in the epidermis and in dermal fibroblasts, as shown in a and b. In addition, distinct signal along the cell-cell junctions within the upper granular cell layer of epidermis is clearly visible, especially in the sections cut on an angle along the epidermis (demonstrated with the double arrows in c and d; the plane of section is highlighted by a dotted line in a and b). Mouse polyclonal antiserum against the periplakin C terminus stains the cell-cell contacts especially strongly in the outer granular cell layer (e). Double labeling with DAPI (f) demonstrates the nuclei. Within the dermis, similar structures stain positive for both periphilin (g) and periplakin (i). Strong apical staining within the epithelial cells lining the excretory ducts of sweat glands (arrows) is demonstrated both in longitudinal and transverse sections. DAPI staining demonstrates the nuclei (h and j), respectively. Tissue sections in a through f were fixed directly in methanol in –20 °C, whereas in g through j, sections were air-dried before fixation, which was found to enhance the signal at the cell periphery but to diminish the nuclear staining.

 

In primary human keratinocytes grown in the elevated Ca2+ concentration (150 µM), periphilin was detected in nuclei, but a signal at the cell-cell contacts was also evident and colocalized with periplakin staining (Fig. 7A). When keratinocytes were exposed to the high Ca2+ concentration (1 mM), periphilin appeared predominately as a diffuse cloudy stain overlapping with periplakin signal, decorating the flattened squames on the top of the keratinocyte layers (Fig. 7B). In the basal keratinocyte layer, periphilin again localized to cell nuclei. Depending on the differentiation stage of the cells in culture, periplakin was also detected as a punctate staining, covering wide areas of flattened cells in culture. A detailed examination of undifferentiated keratinocytes revealed periphilin signal along the nuclear lamina and as a grainy speckle-type staining in the nucleoplasm (Fig. 8A). Sections, obtained with confocal laser microscopy through a cluster of keratinocytes, demonstrated periphilin at the cell periphery throughout the upper keratinocyte layers and revealed the nuclear localization in the basal cell layer (Fig. 8B).



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  		FIG. 7.
Partial colocalization of periplakin and periphilin in keratinocyte cultures. Immunofluorescence microscopy demonstrating keratinocytes double-labeled with periphilin mAb SK30 (A1 and B1) and with rabbit periplakin antiserum 3196 (A2 and B2), together with the DAPI staining (A3 and B3). Human foreskin keratinocytes were grown for 2 days in KGM adjusted to 150 µM Ca2+ (A1–A3) or to 1 mM Ca2+ (B1–B3). Arrowheads point to the nuclear periphilin staining in A1. The colocalization of periphilin and periplakin at the cell-cell junctions is pointed to by arrows in A1 and A2. Differentiated layers of flattened keratinocytes stain strongly for periplakin (B2), whereas periphilin signal is more cloudy and also demonstrates the nuclei of the cells in the basal layer (B1).

 


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  		FIG. 8.
Confocal laser microscopy detects periphilin in multiple subcellular locations. A, fluorescence image demonstrating the nuclear localization of periphilin in proliferating undifferentiated keratinocytes. Localization to the nuclear membrane (arrow 1), to the plane of nuclear cleavage (arrow 2), and in the cytoplasm in a mitotic cell (arrow 3) are pointed out. B, serial optical sections through a differentiated keratinocyte colony (a–g) demonstrate periphilin staining at the cell periphery, especially in the cells on the top of the colony (arrows in b–f and h), whereas a distinct nuclear staining in the basal cell layer is indicated by an arrowhead (g and h). A 20-µm keratinocyte colony was recorded as 0.6-µm horizontal optical sections. Every third section from top to bottom (a–g) and the maximum projection (h) are shown.

 

Overexpression Reveals Cell Type-specific Differences in the Periphilin Subcellular Distribution—A C-terminally Myc-tagged periphilin was expressed in HeLa cells and in Madin-Darby canine kidney (MDCK) cells in a doxycycline-regulatable manner (Fig. 9). A distinct nuclear localization with large and small speckles and thread-like structures was detected in HeLa cells. In mitotic cells, periphilin speckles were distinguishable in the cytoplasm but did not adhere to or colocalize with DNA (Fig. 9A). In MDCK cells, besides a fine, grainy nuclear staining, tread-like aggregates were found to encircle nuclei, extend through the cytoplasm, and traverse across the cell layer connecting the neighboring cells (Fig. 9B).



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  		FIG. 9.
Overexpression of periphilin in cultured cells. Cell lines expressing C-terminally Myc-tagged periphilin under the control of doxycycline regulatable promoter were established from Tet-Off HeLa cells (A) and Tet-Off MDCK cells (B). IIF with Myc tag-specific antibody detected periphilin in HeLa cells in nuclei as fine or coarse granular staining, as pointed to by the arrows in a and b. In mitotic cells (arrowheads in a) periphilin speckles were weak but detectable throughout the cytoplasm. In MDCK cells, both granular nuclear staining (arrow in c) and string-like aggregated filaments (open arrowheads in c, solid arrowheads in d) were detected. Panels a–d show Myc-antibody staining, whereas nuclei are demonstrated through DAPI staining in the corresponding panels a'–d'. Scale bar, 50 µm.

 

Nuclear Localization Correlates with the Insolubility of Periphilin—We also established MDCK cell lines expressing the full-length periphilin (aa 8–374) and periphilin C terminus (aa 197–374) as fusion proteins with GFP. Images of live cells revealed that the GFP-tagged full periphilin accumulated exclusively in the nucleus, leaving the nucleoli empty. The GFP-tagged C terminus (aa 197–374) was found diffusely both in cytoplasm and in the nucleus, resembling the subcellular localization of GFP alone (Fig. 10A). Thus, the N-terminal sequences are responsible for the nuclear localization of periphilin protein. Indeed, a stretch of basis amino acids as a potential candidate for the nuclear localization signal could be detected at the periphilin N terminus, aa 110–115. When cells expressing the GFP-tagged periphilin constructs were extracted with RIPA buffer, and the pellet was further solubilized by boiling in Laemmli-sample buffer, both GFP alone and the GFP-periphilin tail fusion protein became solubilized into the RIPA buffer, but more immunoreactive material was recovered through boiling in SDS-sample buffer (Fig. 10, B and C). In contrast, no signal of the GFP-full periphilin was released by RIPA buffer, and only a weak band was released by boiling in SDS-sample buffer, but a strong signal was detected in the loading well, indicating that most of the GFP-periphilin protein was included in covalently cross-linked macromolecular aggregates that barely entered the gel. In the cells expressing the GFP-full periphilin, a detectable portion of the endogenous periplakin was trapped into the aggregate, which did not enter the gel (Fig. 10D). These results indicate a correlation between the nuclear localization and the insolubility of periphilin protein.



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  		FIG. 10.
Expression of GFP-tagged periphilin in MDCK cells. A, direct fluorescence microscopy of living cells expressing GFP-full periphilin (aa 8–374) (a), GFP-periphilin (aa 197–374) (b), and GFP (c), detected with fluorescein isothiocyanate filter. B, confluent MDCK cells cultures, grown either with (+) or without (–) doxycycline (1 µg/ml), were extracted with RIPA buffer (left panels), and the pellets were solubilized by boiling in SDS-sample buffer (right panels). Proteins were separated on 4–20% SDS-PAGE and immunoblotted with GFP mAb (B), with PHL mAb (C), with periplakin mouse antiserum (D), and with actin mAb. The GFP-full periphilin (aa 8–374) localized into the nuclei (A, panel a) and was not solubilized in RIPA buffer (B and C). Boiling in the SDS-sample buffer released a fraction of that protein, but the major part of the signal did not enter the gel, staying in the well as an insoluble aggregate (B and C). Both GFP-periphilin (aa 197–374) and GFP showed cytoplasmic and nuclear localization (A, panels b and c) and were detected in both the RIPA buffer-soluble and -insoluble fractions (B and C). GFP mAb detected two unspecific bands from all RIPA extracts as well as degradation products of GFP-periphilin (aa 197–374) and GFP (B), which were not detected by PHL mAb (C). The endogenous periplakin was present in each RIPA extract, but a prominent signal was detectable in the insoluble fraction with the full periphilin aggregate (D).

 

Periphilin as a Substrate for Transglutaminase Activity— TGase 2 is widely expressed in cells and tissues. It has been detected in the cytoplasm, in the nucleus and also in the extracellular space as a secreted enzyme, using a wide variety of proteins as substrates for its cross-linking activity (18). Here, a set of bacterially expressed GST-periphilin fusion proteins were tested as substrates for TGase 2 (Fig. 11A). Gel electrophoresis and blotting after incubation with TGase 2 revealed prominent cross-linking and aggregate formation from GST-periphilin fusion proteins, but GST alone was detected as a non-cross-linked monomer. We assumed that endogenous TGases become released when keratinocytes are extracted, and a keratinocyte lysate may be used as an additional source for a cross-linking activity. Indeed, when GST-periphilin fusion proteins, each containing the keratinocyte-specific C terminus, were incubated in the keratinocyte lysate, considerable cross-linking was detected (Fig. 11B). However, the C-terminally deleted periphilin did not serve as a target to the cross-linking activity in the keratinocyte lysate. Duplicate samples supplemented with TGase 2 revealed further aggregation of all three GST-periphilin constructs. Sequential detection of the filter with periplakin antibody showed that the endogenous periplakin was stable in the keratinocyte lysate but was quantitatively cross-linked after addition of TGase 2 into the incubation mixture. The subsequent detection with actin mAb (superimposed in Fig. 11B) showed that TGase 2 slightly reduced the intensity of the actin monomer. GST alone did not become cross-linked with either TGase 2 or with the keratinocyte-specific cross-linking activity.



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  		FIG. 11.
Cross-linking of periphilin by TGase 2 and by an endogenous activity in the keratinocyte lysate. A, recombinant GST-periphilin fusion proteins (200 ng each) were incubated without (–, left panel) or with (+, right panel) TGase 2 (1.0 µg) for 45 min at 37 °C and boiled in the SDS-sample buffer; 40 ng of recombinant protein from each reaction was separated on the 4–20% SDS-PAGE. A, the GST mAb was used for immunoblotting. GST is indicated with an arrowhead, the monomeric GST-periphilin fusion proteins with a bar, and the cross-linked GST-periphilin fusion proteins indicated with the dotted line on the left side of panel A. The TGase 2 treatment resulted in prominent cross-linking of periphilin GST fusion proteins. B, the GST-periphilin fusion proteins were incubated with the keratinocyte lysate (30 µg of protein per reaction) without (–, left panel) or with (+, right panel) TGase 2 (1.0 µg) for 45 min at 37 °C and boiled in the SDS-sample buffer. Proteins were separated on the 4–20% SDS-PAGE, and after the transfer, the filter was sequentially probed with GST mAb, periplakin mAb, and actin mAb. TGase 2 treatment quantitatively cross-linked the endogenous periplakin present in keratinocyte lysate, whereas actin was not cross-linked (arrowheads on the right). Incubation of GST-periphilin fusion proteins, aa 265–374 and aa 197–374, with keratinocyte lysate resulted in the partial cross-linking of these recombinant proteins (dotted line on the right). Addition of TGase 2 further increased the cross-linking. The C-terminally deleted periphilin construct, aa 197–349, was cross-linked by TGase 2 but was resistant to the cross-linking activity in the keratinocyte lysate.

 

Periphilin Is Specifically Cleaved by Caspase-5—Recombinant caspases 1–10 were tested in vitro for their ability to cleave GST-periphilin (aa 197–374) fusion protein. Caspase-5 enzyme, and to a lesser extent caspase-4, produced a distinct 37-kDa cleavage product, which was detected by the GST-specific antibody (Fig. 12). The examination of the periphilin sequence revealed a tetrapeptide LFTD, located between the predicted helixes 1 and 2, which may serve as a recognition sequence for caspase-5. The size of the cleavage product observed on the Western blot agrees with the cleavage to take place after Asp-273. Tetrapeptides with close homology, VFTD in NF-{kappa}B and LQTD in Bcl-2 family protein, are recognized and cleaved by caspase-8 (19). However, caspase-5 is a member of the interleukin-1 converting enzyme family of caspases, which play essential roles in inflammation through cytokine activation, but may also be important mediators of apoptosis (20).



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  		FIG. 12.
Periphilin is specifically cleaved by caspase-5. The GST-periphilin aa 197–374 (A) and, as a control, the GST-periplakin aa 1646–1756 (B), were subjected to the digestion with the recombinant caspases 1–10 (lanes 1–10, respectively). A GST fusion protein with no enzyme addition was included as a control (lane 11). GST, expressed from the pGEX-4T-1 vector is shown in lane 12. The blots were developed with the GST-specific monoclonal antibody. A 37-kDa cleavage product was specifically obtained from GST-periphilin aa 197–374 fusion protein incubated with caspases-1, -4, and -5. The position of the caspase cleavage product is indicated by an arrow, and the GST alone is indicated with the closed arrowhead. The GST-periphilin fusion protein incubated without enzyme addition (lane 11) contained two degradation products, which were not affected by the caspase treatment (lanes 1–10).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We report here the cloning and characterization of periphilin, a novel protein in epidermis. Alternative splicing events give rise to multiple protein isoforms, which are not limited to epidermal keratinocytes. Also, in the EST data base, partial cDNA clones were abundant from a multitude of normal tissues, but also from malignant cells and tissues. However, the C terminus, responsible for the homodimerization and a substrate for cross-linking by an activity in the keratinocyte lysate, is specific for the epidermal isoform of periphilin. The four {alpha}-helixes comprising the C-terminal half of periphilin are reminiscent of the structure of intermediate filament proteins, keratins and lamins. Keratin 18 becomes specifically cleaved during apoptosis within the L1–2 linker between the two {alpha}-helical domains (21, 22). Caspase-6 is thought to function during apoptosis as well as during mitosis in disassembling the nuclear lamina by cleaving lamins A and B at the tetrapeptide within the highly homologous linker L1–2 (23). The group I caspases (1, 4, 5) have been primarily identified as proteases responsible for the proteolytic maturation of proinflammatory cytokines (24). The significance of the observation, that the inflammatory caspases but not the executioner caspases cleave periphilin, remains to be revealed. The caspase recognition site in periphilin is located within the region common to all periphilin isoforms and thus may be used for the processing of periphilin in other cells besides the epidermal keratinocytes.

The monoclonal antibody reported in this work, specific for the periphilin epithelial isoform, detected the protein both in the nucleus and in the cell periphery. An increasing number of components of the plaques of intercellular junctions have been identified also in cell nuclei, suggesting their involvement in nuclear functions and in regulatory interactions between the cell periphery and the nucleus. Besides the cell junction proteins that make transient appearance in the nucleus as a response to various cellular events, plakophilins are characteristic components of desmosomes and occur constitutively in the nucleoplasm of a wide range of cells (25, 26). Plakophilin 2 binds to and potentially regulates {beta}-catenin signaling activity (27). In the nucleus, plakophilin specifically forms complexes with the largest subunit of RNA polymerase III colocalizing into specific nucleoplasmic particles (28). Another dual localization protein, pinin, has been characterized as being associated with the desmosome-intermediate filament complexes (29) but also as a widespread nuclear protein (30) that occurs free in the nucleoplasm and in ribonucleoprotein structures of the speckle type (31), functioning in the pre-mRNA splicing event (32). Symplekin is a multiple localization protein that has been detected within the cytoplasmic plaques of tight junctions (33), but it is a constitutive component in nuclear Cajal bodies and is also present in cytoplasmic particles involved in mRNA biogenesis (34). In epidermis, periphilin localized to the cell-cell junctions only within the upper granular cell layer, where the components of the cornified cell envelope are commonly detected. Periplakin has been detected both associated to the desmosomes (12, 35) and at the interdesmosomal plasma membrane, where the cornified envelope assembly is initiated through cross-linking of involucrin, envoplakin, and periplakin (2). Periphilin may initially be recruited to the plasma membrane through the interaction with periplakin. However, in differentiated keratinocytes in culture, periphilin localization was not limited to that of periplakin. Although periplakin formed a continuous layer extending uniformly along the cell periphery, periphilin was distributed as a cloudy diffuse layer over the apical cell surface, almost resembling the appearance of secreted proteins (36).

In undifferentiated keratinocytes in culture as well as in keratinocytes throughout the epidermis, periphilin was detected as a constitutive nuclear protein. Again, its localization was not limited to a certain compartment, but distinct signal was detected as a grainy staining, in speckle-type interchromosomal structures and lining the nuclear membrane. The distribution pattern closely resembled that of nuclear lamins, which are intermediate filament proteins. In addition to being present in the nuclear lamina, these proteins can also form intranuclear structures associated with sites of DNA replication, and even higher-order tube-like structures have been observed (37, 38).

Besides the specific involvement of the periphilin N terminus in the interaction with periplakin, and the requirement of the extreme C terminus of the epithelial isoform for dimerization, the specific functional role for other domains can only be speculated. The central domain of periphilin showed considerable sequence homology to the aurora-A kinase-like protein, although not to the kinase domain itself. In the case that this region in the kinase is responsible for a specific binding, in an analogous manner, periphilin may be able to associate specifically with components involved in the centrosome-controlled cellular events (39). Although the degree of homology was considerable, the function of the homology domain in each protein remains to be revealed.

The helical domain of periphilin shows structural homology to the conserved motifs that form the reciprocal homodimerization interfaces in vitellogenins and are utilized in microsomal triglyseride transfer protein to form a heterodimer with protein disulfide isomerase (16). In the epidermis, ceramides become covalently cross-linked to proteins, providing the water barrier properties through the assembly of the cornified cell envelope (4). The characteristic insolubility of the cornified cell envelope is largely due to the formation of the isopeptide cross-link between glutamine and lysine residues catalyzed by Ca2+-dependent transglutaminases. It remains to be studied whether periphilin is an authentic component of the cornified cell envelope or if it becomes trapped into that structure in the terminally differentiated keratinocytes. We showed that bacterially expressed C-terminal domain periphilin can function as substrate and become cross-linked by the tissue-type transglutaminase. Periplakin has been detected in soluble and nonsoluble pools in keratinocytes and becomes incorporated into the cornified cell envelope (2, 12, 35). We observed that, although the soluble periplakin in keratinocyte lysate was stable under the incubation conditions, there was an additional cross-linking activity in the keratinocyte lysate that was responsible for producing cross-linked aggregates from the recombinant periphilin domains. However, the C terminus of the epithelial isoform was necessary for the cross-linking with this activity. The addition of TGase 2 further intensified periphilin aggregates, using also the C-terminally deleted construct as a substrate.

Although the GFP-tagged full periphilin was observed to quantitatively localize into the nucleus of the MDCK cells, the GFP-tagged periphilin tail demonstrated diffuse signal throughout the cell, similar to the appearance of GFP alone. Both GFP-tagged periphilin tail and GFP itself were found in the non-ionic detergent-soluble fraction, whereas a fraction of each protein was further solubilized by boiling in the SDS-{beta}-ME Laemmli sample buffer. As a contrast, the GFP-full periphilin was completely insoluble in non-ionic detergents. A considerable portion of this protein was not solubilized by boiling in the SDS-{beta}-ME Laemmli sample buffer either but was detectable in the loading well as covalently cross-linked aggregate. We conclude that the nuclear localization is necessary for the cross-linking of periphilin protein. Although the nuclear localization signal resides within the N-terminal domain, the sequences serving as targets for the cross-linking activity are located within the C-terminal domain of periphilin protein. Among the currently known nine transglutaminases, TGase 2 is a ubiquitously expressed tissue-type enzyme, which has also been identified in the nucleus (40) mediating cross-linking of specific core histone subunits (41).

The expression level of the endogenous periphilin in keratinocytes was not Ca2+-inducible while the subcellular distribution corresponded to the differentiation stage of keratinocytes. Similar to the intermediate filament proteins, periphilin was not soluble in nonionic detergents. However, the sequential extraction with 8 M urea released periphilin polypeptide, and additional polypeptides were detected after boiling in the SDS-{beta}-ME Laemmli sample buffer. Although the nature of the sequences encoded by the alternatively spliced exons was not further addressed in this work, we may speculate differences in solubility properties between the periphilin isoforms. A variety of proteins anchored to membrane lipids are resistant to extraction with nonionic detergents and show considerable stability (42–45). Nuclear matrix proteins are examples of highly insoluble nuclear proteins that form a structural cross-linked framework resembling that of the cytoskeletal intermediate filament network (46, 47).

Based on subcellular localization, cross-linking, insolubility, and the structural similarity to the intermediate filament proteins, lamins and keratins, we conclude that periphilin represents a novel type of structural protein with an ability to function as a general scaffold, providing a stable platform for other proteins to bind to in various subcellular compartments.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY157850 [GenBank] .

* This work was supported by Grant RO1-33588 from NIAMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Dermatology and Cutaneous Biology, Thomas Jefferson University, 233 S. 10th St., BLSB 422, Philadelphia, PA 19107. Tel.: 215-503-2018; Fax: 215-503-5788; E-mail: Sirpa.Aho{at}jefferson.edu.

1 The abbreviations used are: PKB, protein kinase B; MPR, mannose 6-phosphate receptor; aa, amino acid(s); GFP, green fluorescent protein; KGM, keratinocyte growth medium; IIF, indirect immunofluorescence; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DAPI, 4',6-diamidino-2-phenylindole; RT, room temperature; RIPA, radioimmune precipitation assay; TGase, transglutaminase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EST, expressed sequence tag; {beta}-ME, {beta}-mercaptoethanol; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; PHL, periphilin. Back

2 Aho, S. (2003) Cell Tissue Res., in press. Back



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 ABSTRACT
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 DISCUSSION
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