Functional Analysis of the Profilaggrin N-Terminal Peptide: Identification of Domains that Regulate Nuclear and Cytoplasmic Distribution
2002; Elsevier BV; Volume: 119; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.2002.01831.x
ISSN1523-1747
AutoresDavid J. Pearton, Beverly A. Dale, Richard B. Presland,
Tópico(s)Advancements in Transdermal Drug Delivery
ResumoProfilaggrin is expressed in the differentiating granular layer of epidermis and other stratified epithelia, where it forms a major component of cytoplasmic keratohyalin granules. It consists of two distinct domains, an N-terminal S100-like Ca2+- binding domain containing two EF-hands and multiple filaggrin units that aggregate keratin filaments in the stratum corneum. Here, we report structure-function studies of the N-terminal peptide from mouse, human, and rat profilaggrin. The profilaggrin N- terminal peptides of all species contain two S100-like EF-hands, bipartite nuclear localization sequences, and proprotein convertase cleavage sites. The nuclear localization signals in human and mouse profilaggrin were shown to be functional by transfection of epithelial cells and depended on the absence of filaggrin sequences. The nuclear localization of the processed (free) N-terminal peptide of human profilaggrin is consistent with immunolocalization findings in normal human skin and in parakeratotic skin disorders, which exhibit nuclear staining of granular and/or cornified layers. The mouse profilaggrin N-terminus undergoes proteolytic processing in two steps, first releasing an N-terminal peptide containing some filaggrin sequence and finally the free N-terminus of 28–30 kDa; these peptides have cytoplasmic and nuclear distributions, respectively, when expressed in transfected cells. The N-terminal processing may occur prior to or simultaneously with the proteolytic processing of the polyfilaggrin domain. The nuclear accumulation of the profilaggrin N-terminal peptide in epidermis and in transfected cells strongly suggests a calcium-dependent nuclear function for the profilaggrin N-terminus during epidermal terminal differentia tion when the free N-terminus is released from profilaggrin by specific proteolysis. Profilaggrin is expressed in the differentiating granular layer of epidermis and other stratified epithelia, where it forms a major component of cytoplasmic keratohyalin granules. It consists of two distinct domains, an N-terminal S100-like Ca2+- binding domain containing two EF-hands and multiple filaggrin units that aggregate keratin filaments in the stratum corneum. Here, we report structure-function studies of the N-terminal peptide from mouse, human, and rat profilaggrin. The profilaggrin N- terminal peptides of all species contain two S100-like EF-hands, bipartite nuclear localization sequences, and proprotein convertase cleavage sites. The nuclear localization signals in human and mouse profilaggrin were shown to be functional by transfection of epithelial cells and depended on the absence of filaggrin sequences. The nuclear localization of the processed (free) N-terminal peptide of human profilaggrin is consistent with immunolocalization findings in normal human skin and in parakeratotic skin disorders, which exhibit nuclear staining of granular and/or cornified layers. The mouse profilaggrin N-terminus undergoes proteolytic processing in two steps, first releasing an N-terminal peptide containing some filaggrin sequence and finally the free N-terminus of 28–30 kDa; these peptides have cytoplasmic and nuclear distributions, respectively, when expressed in transfected cells. The N-terminal processing may occur prior to or simultaneously with the proteolytic processing of the polyfilaggrin domain. The nuclear accumulation of the profilaggrin N-terminal peptide in epidermis and in transfected cells strongly suggests a calcium-dependent nuclear function for the profilaggrin N-terminus during epidermal terminal differentia tion when the free N-terminus is released from profilaggrin by specific proteolysis. bipartite nuclear localization sequence epidermal differentiation complex green fluorescent protein intermediate filament mouse epidermal keratinocytes proprotein convertase rat epidermal keratinocytes Tris-buffered saline The terminal differentiation of epidermal keratinocytes results in the formation of a mechanically resistant and toughened structure, the stratum corneum, which functions to protect mammals against dessication, and physical and chemical damage. Among the many morphologic changes that occur in normal epidermis during the transition from granular to cornified cells are the dissolution of the nucleus and other organelles, the aggregation of the keratin intermediate filament (IF) network into macrofibrils, and the formation of a cornified cell envelope consisting of loricrin, the small proline-rich proteins, involucrin, and other proteins that are crosslinked by transglutaminases in the stratum corneum (Nemes and Steinert, 1999Nemes Z. Steinert P.M. Bricks and mortar of the epidermal barrier.Exp Mol Med. 1999; 31: 5-19Crossref PubMed Scopus (449) Google Scholar;Presland and Dale, 2000Presland R.B. Dale B.A. Epithelial structural proteins of the skin and oral cavity: function in health and disease.Crit Rev Oral Biol Medical. 2000; 11: 383-408Crossref PubMed Scopus (308) Google Scholar). Although the mechanism of nuclear destruction is not understood, the crosslinking and collapse of keratin IFs into macrofibril bundles is mediated by the IF-associated protein filaggrin, an abundant protein present in granular and transition cells produced by the specific proteolysis of profilaggrin. Profilaggrin is a large, highly phosphorylated, insoluble protein consisting of multiple (10–12 in human, 20 or more in rodents) filaggrin units joined by linker peptides. Profilaggrin is expressed in the granular layer where it is localized within electron-dense nonmembrane bound keratohyalin granules (reviewed inDale et al., 1994Dale B.A. Resing K.A. Presland R.B. Keratohyalin granule proteins.in: Leigh I. Lane B. Watt F. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 323-350Google Scholar;Presland and Dale, 2000Presland R.B. Dale B.A. Epithelial structural proteins of the skin and oral cavity: function in health and disease.Crit Rev Oral Biol Medical. 2000; 11: 383-408Crossref PubMed Scopus (308) Google Scholar). This sequestration into keratohyalin is believed to protect the epithelial cell from the deleterious effects of premature keratin aggregation by filaggrin (Dale et al., 1997Dale B.A. Presland R.B. Lewis S.P. et al.Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.J Invest Dermatol. 1997; 108: 179-187Crossref PubMed Scopus (77) Google Scholar;Kuechle et al., 1999Kuechle M.K. Thulin C.D. Presland R.B. Dale B.A. Profilaggrin requires both linker and filaggrin peptide sequences to form granules: implications for profilaggrin processing in vivo.J Invest Dermatol. 1999; 112: 843-852Crossref PubMed Scopus (33) Google Scholar;Presland et al., 2001Presland R.B. Kuechle M.K. Lewis S.P. et al.Regulated expression of human filaggrin in keratinocytes results in cytoskeletal disruption, loss of cell-cell adhesion, and cell cycle arrest.Exp Cell Res. 2001; 270: 199-213Crossref PubMed Scopus (54) Google Scholar). The profilaggrin N-terminus consists of two distinct domains: a conserved A domain that contains two Ca2+- binding S100-like EF-hands that each bind a Ca2+ ion, and a less conserved cationic B domain (Presland et al., 1992Presland R.B. Haydock P.V. Fleckman P. et al.Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.J Biol Chem. 1992; 267: 23772-23781Abstract Full Text PDF PubMed Google Scholar,Presland et al., 1995Presland R.B. Bassuk J.A. Kimball J.R. Dale B.A. Characterization of two distinct calcium-binding sites in the amino-terminus of human profilaggrin.J Invest Dermatol. 1995; 104: 218-223Crossref PubMed Scopus (52) Google Scholar;Markova et al., 1993Markova N.G. Marekov L.N. Chipev C.C. et al.Profilaggrin is a major epidermal calcium-binding protein.Mol Cell Biol. 1993; 13: 613-625Crossref PubMed Scopus (117) Google Scholar). Profilaggrin belongs to a growing family of high molecular weight proteins with N-terminal S100 domains fused to structurally distinct repetitive regions that are proposed to function as IF-associated proteins and/or cornified envelope components (Fietz et al., 1993Fietz M.J. McLaughlan C.J. Campbell M.T. Rogers G.E. Analysis of the sheep trichohyalin gene: potential structural and calcium-binding roles of trichohyalin in the hair follicle.J Cell Biol. 1993; 121: 855-865Crossref PubMed Scopus (66) Google Scholar;Lee et al., 1993Lee S.C. Kim I.G. Marekov L.N. et al.The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar;Krieg et al., 1997Krieg P. Schuppler M. Koesters R. et al.Repetin (Rptn), a new member of the 'fused gene' subgroup within the S100 gene family encoding a murine epidermal differentiation protein.Genomics. 1997; 43: 339-348Crossref PubMed Scopus (57) Google Scholar;Makino et al., 2001Makino T. Takaishi M. Morohashi M. Huh N.H. Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin.J Biol Chem. 2001; 276: 47445-47452Crossref PubMed Scopus (48) Google Scholar). The genes encoding these "fused S100" proteins are among a large number of epidermally expressed genes that map to human chromosome 1q21 in an ≈1.5 mB region termed the epidermal differentiation complex (EDC) (Marenholz et al., 1996Marenholz I. Volz A. Ziegler A. et al.Genetic analysis of the epidermal differentiation complex (EDC) on human chromosome 1q21: chromosomal orientation, new markers, and a 6-Mb YAC contig.Genomics. 1996; 37: 295-302Crossref PubMed Scopus (103) Google Scholar;Mischke et al., 1996Mischke D. Korge B.P. Marenholz I. et al.Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ('epidermal differentiation complex') on human chromosome 1q21.J Invest Dermatol. 1996; 106: 989-992Crossref PubMed Scopus (421) Google Scholar;South et al., 1999South A.P. Cabral A. Ives J.H. et al.Human epidermal differentiation complex in a single 2.5 Mbp long continuum of overlapping DNA cloned in bacteria integrating physical and transcript maps.J Invest Dermatol. 1999; 112: 910-918Crossref PubMed Scopus (59) Google Scholar). An equivalent EDC is present on mouse chromosome 3 (Song et al., 1999Song H.J. Poy G. Darwiche N. et al.Mouse Sprr2 genes: a clustered family of genes showing differential expression in epithelial tissues.Genomics. 1999; 55: 28-42Crossref PubMed Scopus (63) Google Scholar). During terminal differentiation profilaggrin is processed by several endoproteases to generate two major products: filaggrin and the N-terminal peptide. Profilaggrin processing to filaggrin requires both the removal of multiple phosphates from each filaggrin unit within profilaggrin by phosphatases and its specific proteolysis by a number of proteases (Lonsdale-Eccles et al., 1982Lonsdale-Eccles J.D. Teller D.C. Dale B.A. Characterization of a phosphorylated form of the intermediate filament-aggregating protein filaggrin.Biochemistry. 1982; 21: 5940-5948Crossref PubMed Scopus (48) Google Scholar;Harding and Scott, 1983Harding C.R. Scott I.R. Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.J Mol Biol. 1983; 170: 651-673Crossref PubMed Scopus (200) Google Scholar;Resing et al., 1989Resing K.A. Walsh K.A. Haugen-Scofield J. Dale B.A. Identification of proteolytic cleavage sites in the conversion of profilaggrin to filaggrin in mammalian epidermis.J Biol Chem. 1989; 264: 1837-1845Abstract Full Text PDF PubMed Google Scholar;Kam et al., 1993Kam E. Resing K.A. Lim S.K. Dale B.A. Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2A family.J Cell Sci. 1993; 106: 219-226PubMed Google Scholar). The proteases that are involved in profilaggrin processing include profilaggrin endoproteinase 1 (PEP1), a chymotrypsin-like protease (Resing et al., 1995bResing K.A. Thulin C. Whiting K. et al.Characterization of profilaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase of epidermis.J Biol Chem. 1995; 270: 28193-28198Crossref PubMed Scopus (49) Google Scholar), calpain (Resing et al., 1993aResing K.A. Al-Alawi N. Blomquist C. et al.Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.J Biol Chem. 1993; 268: 25139-25145Abstract Full Text PDF PubMed Google Scholar;Yamazaki et al., 1997Yamazaki M. Ishidoh K. Suga Y. et al.Cytoplasmic processing of human profilaggrin by active mu-calpain.Biochem Biophys Res Commun. 1997; 235: 652-656Crossref PubMed Scopus (50) Google Scholar), and furin, a member of the proprotein convertase (PC) family of endoproteases (Pearton et al., 2001Pearton D.J. Nirunsuksiri W. Rehemtulla A. et al.Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates.Exp Dermatol. 2001; 10: 193-203Crossref PubMed Scopus (80) Google Scholar). The order of these processing events has not been elucidated; however, as profilaggrin intermediates containing two or more filaggrin units lack detectable phosphate it is thought that most dephosphorylation occurs before processing of the polyfilaggrin domain occurs (Harding and Scott, 1983Harding C.R. Scott I.R. Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.J Mol Biol. 1983; 170: 651-673Crossref PubMed Scopus (200) Google Scholar). Filaggrin binds to cytoplasmic keratin IFs to form the macrofibrils that are retained in cornified cells, whereas the N-terminal peptide localizes to nuclei of epidermal granular and transition cells (Ishida-Yamamoto et al., 1998Ishida-Yamamoto A. Takahashi H. Presland R.B. et al.Translocation of profilaggrin N-terminal domain into keratinocyte nuclei with fragmented DNA in normal human skin and loricrin keratoderma.Laboratory Invest. 1998; 78: 1245-1253PubMed Google Scholar;Presland and Dale, 2000Presland R.B. Dale B.A. Epithelial structural proteins of the skin and oral cavity: function in health and disease.Crit Rev Oral Biol Medical. 2000; 11: 383-408Crossref PubMed Scopus (308) Google Scholar). The mechanism and function of this nuclear translocation is unclear. In loricrin keratoderma, a disease affecting palm and sole skin characterized by parakeratosis in the lower cornified layers, the profilaggrin N-terminus is associated with nuclear aggregates of a mutant loricrin protein that might prevent it from functioning normally (Ishida-Yamamoto and Iizuka, 1998Ishida-Yamamoto A. Iizuka H. Structural organization of cornified cell envelopes and alterations in inherited skin disorders.Exp Dermatol. 1998; 7: 1-10Crossref PubMed Scopus (107) Google Scholar;Ishida-Yamamoto et al., 1998Ishida-Yamamoto A. Takahashi H. Presland R.B. et al.Translocation of profilaggrin N-terminal domain into keratinocyte nuclei with fragmented DNA in normal human skin and loricrin keratoderma.Laboratory Invest. 1998; 78: 1245-1253PubMed Google Scholar). Studies of affected patients and transgenic mice that are either loricrin-deficient and/or express the same mutant protein have shown that the loricrin mutation is pathogenic, and that the resulting skin disease is probably due to disruption of nuclear events during terminal differentiation rather than to an absence of loricrin from cornified envelopes (Ishida-Yamamoto et al., 2000Ishida-Yamamoto A. Kato H. Kiyama H. et al.Mutant loricrin is not crosslinked into the cornified cell envelope but is translocated into the nucleus in loricrin keratoderma.J Invest Dermatol. 2000; 115: 1088-1094Crossref PubMed Scopus (34) Google Scholar;Koch et al., 2000Koch P.J. de Viragh P.A. Scharer E. et al.Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein.J Cell Biol. 2000; 151: 389-400Crossref PubMed Scopus (228) Google Scholar;Suga et al., 2000Suga Y. Jarnik M. Attar P.S. et al.Transgenic mice expressing a mutant form of loricrin reveal the molecular basis of the skin diseases, Vohwinkel syndrome and progressive symmetric erythrokeratoderma.J Cell Biol. 2000; 151: 401-412Crossref PubMed Scopus (62) Google Scholar). In this paper, we report the sequence of the mouse and rat profilaggrin N-termini and show its similarity to the human profilaggrin N-terminus in terms of both structure and proteolytic processing in keratinocytes. Like its human counterpart, the mouse N-terminal peptide is specifically cleaved in vivo during terminal differentiation. We show that the cationic B domain of the profilaggrin N-terminus contains functional bipartite nuclear localization sequence(s) (bNLSs) that facilitate nuclear accumulation in keratinocytes. Removal of filaggrin sequences by proteolytic cleavage, however, is required for this nuclear translocation. Profilaggrin processing therefore provides a potential link between two major events of terminal differentiation: the aggregation of keratin filaments and nuclear dissolution. A 129/SvJ mouse genomic BAC library (Genome Systems, St. Louis, MO) was screened with a pair of polymerase chain reaction (PCR) primers specific for the mouse profilaggrin 3′ noncoding sequence. BAC plasmid DNA was isolated from overnight cultures of each clone using Nucleobond AX columns (The Nest Group, Southboro, MA). The profilaggrin gene was localized within BAC clones by southern blot hybridization of restriction digests with coding and 3′ noncoding filaggrin DNA probes labeled with [α-32P]-dCTP (3000 Ci per mmol) (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York1989Google Scholar). Several restriction endonuclease fragments that hybridized with coding probes were subcloned into pGEM5Zf+ and pGEM7Zf+ vectors (Promega, Madison, WI) and sequenced to derive the 5′ end of the mouse profilaggrin coding sequence. To confirm the coding sequence and exon/intron organization of the mouse profilaggrin gene, the cDNA sequence was isolated by reverse transcription PCR (RT-PCR) of mouse epidermal RNA as described previously (Presland et al., 2000Presland R.B. Boggess D. Lewis S.P. et al.Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.J Invest Dermatol. 2000; 115: 1072-1081Crossref PubMed Scopus (152) Google Scholar) using the following primers derived from the genomic sequence: 5′-GTGCATACACACTACTA (upstream exon 2 primer) and 5′-AGAAAGTAGTAGGCATG (downstream exon 3 primer). The cDNA product (≈1 kb) containing the N-terminal domain of mouse profilaggrin was cloned into TOPO-TA vector (Invitrogen, Carlsbad, CA) for sequence analysis. For rat profilaggrin, genomic and cDNA sequences were obtained by PCR amplification of DNA and RNA isolated from rat epidermal keratinocytes (REKs) (Baden and Kubilus, 1983Baden H.P. Kubilus J. The growth and differentiation of cultured newborn rat keratinocytes.J Invest Dermatol. 1983; 80: 124-130Crossref PubMed Scopus (80) Google Scholar;Haydock et al., 1993Haydock P.V. Blomquist C. Brumbaugh S. et al.Antisense profilaggrin RNA delays and decreases profilaggrin expression and alters in vitro differentiation of rat epidermal keratinocytes.J Invest Dermatol. 1993; 101: 118-126Abstract Full Text PDF PubMed Google Scholar). Genomic DNA was isolated using a Qiamp tissue kit (Qiagen, Chatsworth, CA). For genomic DNA amplifications, PCR assays (32 cycles at 94°C for 50 s, 47.5°C for 60 s, and 72°C for 90 s) were performed using Taq polymerase (Promega, Madison, WI) in the presence of 5% dimethylsulfoxide and 2.5 mM MgCl2. The 5′ primer corresponded to nucleotides 1–24 of the coding region of the human profilaggrin sequence (5′-ATGTCTACTCTCCTGGAAAACATC-3′; GenBank accession L01088). The 3′ primer was directed against the linker region of the rat filaggrin sequence (5′-GGACTC GTCCTCGGTTTCTTCTACACC-3′; GenBank accession M21759). The resulting 2.2 kb PCR products were cloned into pCR2.1 TA (Invitrogen) for sequencing. Sequence analysis of both mouse and rat profilaggrin clones was performed by dye terminator cycle sequencing using the BigDye kit (Perkin Elmer ABI, Foster City, CA). To obtain the entire sequence for both strands of mouse and rat profilaggrin, internal primers were synthesized and the resultant overlapping sequences were assembled using either the Sequencher program (Gene Codes, Ann Arbor, MI) or PC/GENE. To determine the exon/intron boundaries of the rat profilaggrin sequence, total RNA was extracted from confluent REKs and single stranded cDNA was prepared as described previously (Presland et al., 2001Presland R.B. Kuechle M.K. Lewis S.P. et al.Regulated expression of human filaggrin in keratinocytes results in cytoskeletal disruption, loss of cell-cell adhesion, and cell cycle arrest.Exp Cell Res. 2001; 270: 199-213Crossref PubMed Scopus (54) Google Scholar). PCR was performed with the primers 5′-CACTAG CATGATTGA CATATTCC-3′ and 5′-TTGAAGTCTGCCCTT GCCCG-3′, using 32 cycles of 94°C for 50 s, 53°C for 45 s, and 72°C for 50 s in the presence of Taq polymerase (Promega) and Taq extender (Stratagene, La Jolla, CA). The PCR product (1078 bp) was cloned into pCR2.1 (Invitrogen) and sequenced. A partial genomic sequence was also obtained from Sprague-Dawley rats that express a small 350 kDa variant of profilaggrin containing 7–9 filaggrin units (K. Resing, personal communication) using the 5′ primerCATGCAAATGCTAGATGTGG-3′ and the same 3′ primer used above. The N-terminal sequence of rat profilaggrin protein was verified by Endolys C digestion of purified rat profilaggrin followed by matrix assisted laser desorption ionization mass spectrometry and electrospray ionization mass spectrometry (Resing et al., 1993bResing K.A. Johnson R.S. Walsh K.A. Characterization of protease processing sites during conversion of rat profilaggrin to filaggrin.Biochemistry. 1993; 32: 10036-10045Crossref PubMed Scopus (47) Google Scholar;Resing et al., 1995aResing K.A. Johnson R.S. Walsh K.A. Mass spectrometric analysis of 21 phosphorylation sites in the internal repeat of rat profilaggrin, precursor of an intermediate filament associated protein.Biochemistry. 1995; 34: 9477-9487Crossref PubMed Scopus (72) Google Scholar) to be STLLESITSMIDIFQQYSNNDK (K. Resing, personal communication). This sequence exhibits four amino acid differences from mouse and nine from human profilaggrin (see Figure 1b ). Sequence alignments were performed using the programs NALIGN (PC/GENE) and CLUSTALW (PBIL) and outputted with BOXSHADE. Peptide motifs were identified using the programs PROSITE (http://www.expasy.org/prosite) and PSORT II (http://www.psort.nibb.ac.jp). A polyclonal antipeptide antibody, Am1, was developed against a 15 amino acid sequence (E38GQLQAVLKNPDDQD53) in the region between the EF-hands of the mouse A domain (see Figure 1b) (Genemed Synthesis, San Francisco, CA). This sequence is equivalent to the peptide used to prepare the human-A-domain-specific antibody (Presland et al., 1997Presland R.B. Kimball J.R. Kautsky M.B. et al.Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.J Invest Dermatol. 1997; 108: 170-178Crossref PubMed Scopus (73) Google Scholar). The antibodies were affinity purified prior to use. Cell or tissue extracts were prepared in urea-Tris buffer as previously described (Presland et al., 1997Presland R.B. Kimball J.R. Kautsky M.B. et al.Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.J Invest Dermatol. 1997; 108: 170-178Crossref PubMed Scopus (73) Google Scholar), or in a modified RIPA buffer containing 1% Nonidet P-40 (Pearton et al., 2001Pearton D.J. Nirunsuksiri W. Rehemtulla A. et al.Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates.Exp Dermatol. 2001; 10: 193-203Crossref PubMed Scopus (80) Google Scholar). For immunoblotting, equal protein loadings were resolved on either 7%-12% or 10%-15% gradient sodium dodecyl sulfate (SDS) polyacrylamide gels and electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH). After blocking with 2% nonfat dry milk powder the membranes were probed with primary antibody and detected with a horseradish-peroxidase-conjugated donkey antirabbit IgG (Amersham, Arlington Heights, IL) using the Renaissance chemiluminescent substrate (NEN, Boston, MA). Mouse epidermal keratinocytes (MEKs) were cultured in low (0.06 mM) Ca2+ NIH-3T3 fibroblast conditioned medium as described previously (Hager et al., 1999Hager B. Bickenbach J.R. Fleckman P. Long-term culture of murine epidermal keratinocytes.J Invest Dermatol. 1999; 112: 971-976Crossref PubMed Scopus (107) Google Scholar). Cells were passaged and seeded at low density (2.4 × 104 cells per cm2) and reached confluence in 7 d. Confluent cultures were fed with 3T3-conditioned medium every 48 h and the detached cells were harvested by centrifugation of the culture medium. The cell pellet was washed with cold Dulbecco's phosphate-buffered saline (PBS) before extraction. Adherent cells were washed twice with cold PBS before being scraped from the dish and extracted in urea-Tris or modified RIPA buffer. Unextracted cell pellets and cell extracts were stored at -80°C until use. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) and equal protein loads from each pellet were loaded onto SDS polyacrylamide gels and blotted onto nitrocellulose for Western blot detection. The phosphorylation state of profilaggrin and its processing products was determined by labeling confluent keratinocytes with radiolabeled phosphate. Two day postconfluent MEKs were grown in the presence of 100 μCi [32P]-orthophosphate for 24 h. Labeled cells were harvested by centrifugation of the medium (for detached cells) or scraping the dish surface (for attached cells). After washing away unbound orthophosphate in cold PBS, cells were extracted in urea-Tris or RIPA buffer. The RIPA buffer extract was subjected to immunoprecipitation using either antimouse profilaggrin antibody (Covance, Richmond, CA) or the Am1 antibody. Antibody-antigen complexes were recovered using protein-A-coated paramagnetic beads (Dynal, Lake Pleasant, NY), eluted with 0.1 M glycine pH 2.8 or by heating at 45°C for 15 min in 2% SDS Laemmli loading buffer. The extracts and immunoprecipitates were run on 7.5%-12% SDS polyacrylamide gels and either blotted onto nitrocellulose for Western analysis or dried and exposed to X-ray film. Immunofluorescence was per formed on frozen or methyl Carnoys fixed tissue samples embedded in paraffin (Presland et al., 1997Presland R.B. Kimball J.R. Kautsky M.B. et al.Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.J Invest Dermatol. 1997; 108: 170-178Crossref PubMed Scopus (73) Google Scholar) or transfected cells grown on glass coverslips fixed with ice-cold methanol:acetone (3:1) (Dale et al., 1997Dale B.A. Presland R.B. Lewis S.P. et al.Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.J Invest Dermatol. 1997; 108: 179-187Crossref PubMed Scopus (77) Google Scholar). After rehydration (for Carnoys fixed tissues) sections were washed in Tris-buffered saline (TBS) and processed for single or double immunofluorescence. Sections were incubated with primary antibody overnight at 4°C in TBS containing 1% bovine serum albumin. The primary antibodies were detected using either fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG (Vector Laboratories, Burlingame, CA) or biotinylated goat antirabbit IgG followed by streptavidin conjugated to Texas Red (Vector Laboratories). For detection of mouse filaggrin a directly labeled FITC-antifilaggrin rabbit IgG (Covance) was used after the initial secondary was removed. Multiple rinses in TBS were done between each step. For DNA staining, samples were incubated in 0.001% 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, St. Louis, MO) for 10 min and then washed with water. Samples were air-dried and coverslipped using Prolong mounting medium (Molecular Probes, Eugene, OR) to minimize fading. All the samples were observed under an epifluorescence Nikon-SA Microphot microscope and digital images were collected using a CCD camera (Photometrics, Tucson, AZ). DNA constructs were prepared from mouse and human profilaggrin cDNA clones (Presland et al., 1997Presland R.B. Kimball J.R. Kautsky M.B. et al.Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.J Invest Dermatol. 1997; 108: 170-178Crossref PubMed Scopus (73) Google Scholar; this paper) by a PCR approach followed by cloning into NT-GFP-TOPO (Invitrogen) to generate constructs with an N-terminal green fluorescent protein (GFP) tag. The primers used for cloning the complete mouse profilaggrin N-terminus were 5′-TCCGCTCTC CTGGAAAGCATC-3′ (MP-5) and 5′-TTAGCCTGCCCTGGATCT CCTCTG-3′ (MP-3); the human primers were 5′-TCTACTCTC CTGGAAAACATCTTTG-3′ (HP-5) and 5′-TTACCTACGCTTTCTTGTCCTGG-3′ (HP-3) (termination codons engineered at the 3′ end of each construct are underlined). The human profilaggrin N-terminal mutant and deletion constructs were prepare
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