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Effect of Mutation and Phosphorylation of Type I Keratins on Their Caspase-mediated Degradation

2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês

10.1074/jbc.m103315200

1083-351X

Nam‐On Ku, M. Bishr Omary,

Wnt/β-catenin signaling in development and cancer

Type I keratins K18 and K19 undergo caspase-mediated degradation during apoptosis. Two known K18 caspase cleavage sites are aspartates in the consensus sequences VEVDA and DALDS, located within the rod domain and tail domain, respectively. Several K14 (another type I keratin) mutations within the caspase cleavage motif have been described in patients with epidermolysis bullosa simplex. Here we use extensive mutational analysis to show that K19 and K14 are caspase substrates and that the ability to undergo caspase-mediated digestion of K18, K19, or K14 is highly dependent on the location and nature of the mutation within the caspase cleavage motif. Caspase cleavage of K14 occurs at the aspartate of VEMDA, a consensus sequence found in type I keratins K12–17 with similar but not identical sequences in K18 and K19. For K18, apoptosis-induced cleavage occurs sequentially, first at 393DALD and then at 234VEVD. Hyperphosphorylation of K18 protects from caspase-3 in vitro digestion at 234VEVD but not at 393DALD. Hence, keratins K12–17 are likely caspase substrates during apoptosis. Keratin hyperphosphorylation, which occurs early in apoptosis, protects from caspase-mediated K18 digestion in a cleavage site-specific manner. Mutations in epidermolysis bullosa simplex patients could interfere with K14 degradation during apoptosis, depending on their location. Type I keratins K18 and K19 undergo caspase-mediated degradation during apoptosis. Two known K18 caspase cleavage sites are aspartates in the consensus sequences VEVDA and DALDS, located within the rod domain and tail domain, respectively. Several K14 (another type I keratin) mutations within the caspase cleavage motif have been described in patients with epidermolysis bullosa simplex. Here we use extensive mutational analysis to show that K19 and K14 are caspase substrates and that the ability to undergo caspase-mediated digestion of K18, K19, or K14 is highly dependent on the location and nature of the mutation within the caspase cleavage motif. Caspase cleavage of K14 occurs at the aspartate of VEMDA, a consensus sequence found in type I keratins K12–17 with similar but not identical sequences in K18 and K19. For K18, apoptosis-induced cleavage occurs sequentially, first at 393DALD and then at 234VEVD. Hyperphosphorylation of K18 protects from caspase-3 in vitro digestion at 234VEVD but not at 393DALD. Hence, keratins K12–17 are likely caspase substrates during apoptosis. Keratin hyperphosphorylation, which occurs early in apoptosis, protects from caspase-mediated K18 digestion in a cleavage site-specific manner. Mutations in epidermolysis bullosa simplex patients could interfere with K14 degradation during apoptosis, depending on their location. intermediate filament antibody anisomycin t-butoxycarbonyl-Asp·(O-methyl)-fluoromethyl ketone epidermolysis bullosa simplex monoclonal antibody polyacrylamide gel electrophoresis wild-type linker 1–2 Keratins are the cytoplasmic intermediate filament (IF)1 proteins of epithelial cells and consist of >20 separate gene products (1Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1284) Google Scholar). The keratin subfamily of IF proteins is classified into two major groups, type I keratins (K9–20) and type II keratins (K1–8), which associate as noncovalent type I-II heteropolymers in an epithelial cell type-specific manner (1Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1284) Google Scholar, 2Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4541) Google Scholar, 3Ku N.O. Zhou X. Toivola D.M. Omary M.B. Am. J. Physiol. 1999; 277: G1108-G1137PubMed Google Scholar, 4Herrmann H. Aebi U. Curr. Opin. Cell Biol. 2000; 12: 79-90Crossref PubMed Scopus (420) Google Scholar). Among cytoplasmic IF proteins, keratins and vimentin undergo caspase-mediated degradation as part of the cytoskeletal remodeling that takes place during apoptosis (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar, 6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 7Leers M.P. Kolgen W. Bjorklund V. Bergman T. Tribbick G. Persson B. Bjorklund P. Ramaekers F.C. Bjorklund B. Nap M. Jornvall H. Schutte B. J. Pathol. 1999; 187: 567-572Crossref PubMed Scopus (573) Google Scholar, 8Prasad S. Soldatenkov V.A. Srinivasarao G. Dritschilo A. Int. J. Oncol. 1999; 14: 563-570PubMed Google Scholar). The nuclear lamin IF proteins also undergo degradation during apoptosis and were the first IF proteins demonstrated to undergo apoptosis-associated digestion (9Lazebnik Y.A. Takahashi A. Moir R.D. Goldman R.D. Poirier G.G. Kaufmann S.H. Earnshaw W.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9042-9046Crossref PubMed Scopus (483) Google Scholar, 10Takahashi A. Alnemri E.S. Lazebnik Y.A. Fernandes-Alnemri T. Litwack G. Moir R.D. Goldman R.D. Poirier G.G. Kaufmann S.H. Earnshaw W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8395-8400Crossref PubMed Scopus (472) Google Scholar, 11Rao L. Perez D. White E. J. Cell Biol. 1996; 135: 1441-1455Crossref PubMed Scopus (513) Google Scholar). The only keratins shown to undergo proteolysis during apoptosis are K18 and K19, whereas their type II partner (i.e. K8) manifests remarkable resistance to apoptotic degradation. Two known K18 caspase sites, VEVD and DALD, are located in the rod domain and tail domain, respectively (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar, 6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 7Leers M.P. Kolgen W. Bjorklund V. Bergman T. Tribbick G. Persson B. Bjorklund P. Ramaekers F.C. Bjorklund B. Nap M. Jornvall H. Schutte B. J. Pathol. 1999; 187: 567-572Crossref PubMed Scopus (573) Google Scholar). VEVD or similar consensus sequences are found in other IF proteins within the so-called linker 1–2 (L1–2) region of the rod domain (Fig. 1), whereas DALD is a unique caspase site that is found only in the K18 tail domain. The signals, if any, that target keratin degradation and the significance of this proteolysis are unknown. To that end, the only keratin-related apoptosis-associated change after an apoptotic signal is marked early keratin hyperphosphorylation. The significance of this early keratin hyperphosphorylation in association with apoptosis is not known, but amino acid substitution of the major K18 phosphorylation sites does not alter susceptibility to caspase digestion (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Understanding the significance and regulation of keratin (and other IF protein) degradation during apoptosis is important from a cell biological perspective and may also have pathophysiological relevance to human disease. For example, although most keratin mutations that have been identified in patients with epidermal blistering keratin diseases are located at the N-terminal region of the rod IA subdomain (12Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (836) Google Scholar, 13Irvine A.D. McLean W.H. Br. J. Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (329) Google Scholar), at least four K14 mutations have been described within the L1–2 region (14Rugg E.L. Morley S.M. Smith F.J. Boxer M. Tidman M.J. Navsaria H. Leigh I.M. Lane E.B. Nat. Genet. 1993; 5: 294-300Crossref PubMed Scopus (80) Google Scholar, 15Humphries M.M. Sheils D.M. Farrar G.J. Kumar-Singh R. Kenna P.F. Mansergh F.C. Jordan S.A. Young M. Humphries P. Hum. Mutat. 1993; 2: 37-42Crossref PubMed Scopus (53) Google Scholar, 16Chen H. Bonifas J.M. Matsumura K. Ikeda S. Leyden W.A. Epstein Jr., E.H. J. Invest. Dermatol. 1995; 105: 629-632Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 17Muller F.B. Kuster W. Bruckner-Tuderman L. Korge B.P. J. Invest. Dermatol. 1998; 111: 900-902Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) in close proximity to the caspase recognition motif (VEMDA, also referred to herein as the caspase box). The cause of blister formation in these patients may be attributed to keratin filament assembly defects with resultant cell fragility. However, the proximity of these mutations to the caspase digestion site raises the possibility that the phenotype of the keratin disease in these instances may also be associated with perturbations in keratin degradation. If so, this could potentially impact the disease pathophysiology in patients with epidermolysis bullosa simplex (EBS), who harbor K14 L1–2 region mutations, and may offer more directed therapeutic strategies. In this study, we use a mutagenesis approach to confirm that Asp396 in the K18 tail domain is a caspase cleavage site in vivo. In addition, we show that sequential K18 digestion occurs at the tail (DALDS) and then at the rod (VEVDA) domains and that keratin hyperphosphorylation protects against cleavage at the rod but not the tail motif. To address the significance of the caspase box residues in keratin degradation, we generated a battery of caspase box mutations that mimicked the K14 mutations described in EBS patients and examined parallel mutations in K18 and K19. The results show that: (i) K14 VEMDA→MEMDA or VEMDD has no effect on susceptibility to caspase digestion, (ii) K14 VEMDA→VERDA and equivalent K18 and K19 mutations altered the migration of the N-terminal fragment on gel analysis, and (iii) K14 VEMDA→VEMGA and the equivalent K18 mutation abolished caspase digestion (boldface letters indicate residues that are mutated). Hence, pathogenic K14 mutations within the L1–2 region in patients with EBS can prevent caspase-mediated keratin degradation during apoptosis. In addition, the VEMDA caspase box, which is found in many type I keratins (K12–17) is a suitable caspase substrate, as shown here for K14. The primary monoclonal antibodies (mAbs) used (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) were L2A1 (anti-K8/K18), D5 (anti-K18), DC10 (anti-K18; Neomakers, Fremont, CA), M30 (anti-K18 Asp396 fragment;Roche Molecular Biochemicals), and KA4 (anti-K19). Other reagents and antibodies used were rabbit anti-K14 (ICN Biomedicals, Inc., Aurora, OH), anisomycin (An), caspase inhibitor III (t-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone (BOC)), human recombinant caspase-3 (Calbiochem, La Jolla, CA), and calf intestine alkaline phosphatase (Roche Molecular Biochemicals). HT29 (human colon) and BHK-21 (hamster kidney) cells (American Type Culture Collection, Manassas, VA) were cultured in media as recommended by the supplier. HT29 cells express K8, K18, and K19, whereas BHK-21 cells do not express any easily detectable keratins (data not shown). Wild-type (WT) or mutant keratin constructs, generated using a TransformerTM kit (CLONTECH), were transfected using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. Notably, this transfection method induces apoptosis and keratin degradation in BHK-21 cells as reported previously (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Keratin degradation and subsequent apoptosis were induced in HT29 cells by culturing the cells in the presence of An (10 µg/ml in Me2SO) for 0, 0.5, 1, 2, 4, 8, 12, or 16 h. Total cell lysates in 2% SDS-containing sample buffer were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and then transferred to polyvinylidene difluoride membranes, followed by immunoblotting (19Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). BHK-21 cells transiently transfected with K8 and/or K18 were treated with 0.1% Me2SO or 50 µm caspase inhibitor III (BOC) overnight, followed by isolation of total cell lysates and immunoblotting. HT29 cells were solubilized with 1% Nonidet P-40 in phosphate-buffered saline containing a mixture of protease inhibitors (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). After 1 h, lysates were pelleted, and the supernatant was used for immunoprecipitation by incubation in the presence of Sepharose-protein A coupled to mAb L2A1. Two duplicate K8/K18 immunoprecipitates were either used as a control or incubated with calf intestine alkaline phosphatase (20 units) for 1 h at room temperature to obtain dephosphorylated K8/K18. Hyperphosphorylated K8/K18 immunoprecipitates were obtained from HT29 cells treated with 1 µg/ml okadaic acid for 2 h. The immunoprecipitates (control, dephosphorylated, or hyperphosphorylated K8/K18) were incubated with buffer alone or with buffer containing recombinant human caspase-3 for 0.5, 1.5, or 3 h. The samples were then separated by SDS-PAGE and analyzed by immunoblotting. Previous studies utilizing K18 D237A (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar) or direct sequencing of apoptosis-generated K18 fragment b (Fig.1 and Ref. 6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) showed that K18 Asp237 is a major cleavage site in vivo. In addition, in vitro digestion of K18 using caspase-3 or caspase-7 indicated that a second caspase site is likely to be present in the tail domain of K18 (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar). The second site was inferred to be K18 Asp396 by epitope mapping using mAb M30 (7Leers M.P. Kolgen W. Bjorklund V. Bergman T. Tribbick G. Persson B. Bjorklund P. Ramaekers F.C. Bjorklund B. Nap M. Jornvall H. Schutte B. J. Pathol. 1999; 187: 567-572Crossref PubMed Scopus (573) Google Scholar), although this was not formally tested by mutational analysis. We mutated K18 D396E and K18 D237/396E and examined caspase digestion of the mutants as compared with WT K18-transfected BHK-21 cells undergoing apoptosis. K18 D396E generated a "new" 27-kDa band (K18 b+c fragment) that was recognized by a K18 C-terminal-specific Ab (Fig. 1 A; Fig.2, A and C), whereas K18 D237E accumulated a 43-kDa band (K18 a+b fragment; Fig.2 A) that was recognized by a K18 N-terminal-specific Ab (Fig. 1 A; Fig. 2, B and D). The double mutant K18 D237/396E generated only one major undigested K18 species (Fig. 2 A, lane 5). Hence, both K18 Asp237 and Asp396 are caspase digestion sites in vivo. Generation of the K18 fragments a+b, a, or b was inhibited using the broad range caspase inhibitor BOC in cells transfected with WT K18 or with WT K8/K18 (Fig. 3 A), thereby indicating that K18 fragmentation at Asp237 and Asp396 is caspase-mediated. Antibody specificity was confirmed by blotting K8-transfected cells (Fig. 3 A; K8 expression was determined by blotting with anti-K8-specific Ab; data not shown). The M30 mAb (which recognizes an epitope that becomes exposed after K18 is cut at Asp396) does not detect K18 when mutated at D396E or D237/396E (Fig. 3 B, lanes 4 and5) as predicted from its reactivity with K18 synthetic peptides (7Leers M.P. Kolgen W. Bjorklund V. Bergman T. Tribbick G. Persson B. Bjorklund P. Ramaekers F.C. Bjorklund B. Nap M. Jornvall H. Schutte B. J. Pathol. 1999; 187: 567-572Crossref PubMed Scopus (573) Google Scholar). In addition, M30 reactivity for K18 a+b fragment increases dramatically in K18 D237E as compared with WT K18 transfectants, because K18 a+b fragment accumulates because it can no longer be further degraded (Fig. 3 B, compare lanes 2 and 3). We then tested, using An-induced apoptosis, whether caspase digestion occurs sequentially or randomly at VEVD and DALD. Immunoblotting of total lysates from HT29 cells treated with An for various time intervals with M30 showed that K18 a+b (i.e. digestion at DALD) begins to appear after 0.5 h, whereas K18 b (i.e.digestion at VEVD) is detected after 2 h of An treatment (Fig.3 C). This indicates that upon An-induced apoptosis, Asp396 is initially cleaved followed by digestion at Asp237. Cleavage at Asp396 does not appear to be essential for cleavage at Asp237 because the K18 D396E mutant remains a caspase substrate at Asp237 (Fig. 2, compare lanes 2 and 4). We showed previously that K8 (Ser73 and Ser431) and K18 (Ser52) but not K18 (Ser33) hyperphosphorylation occurred within 0.5 h after An treatment and that keratin Ser→Ala mutants at these sites remain comparable to WT K18 in terms of their susceptibility to caspase digestion (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Here, we examined the effect of dephosphorylation or hyperphosphorylation on K18 fragmentation using in vitrodigestion by caspase-3. K8/K18 immunoprecipitates that were isolated from okadaic acid-treated cells or treated with alkaline phosphatase were digested with caspase-3 in vitro and then analyzed for the formation of keratin fragments. As shown in Fig.4 A, K18 hyperphosphorylation inhibited digestion at K18 Asp237 without any significant effect on K18 digestion at Asp396. In contrast, dephosphorylation did not have a significant effect on K18 Asp237 or Asp396 digestion (Fig.4 A), which is consistent with our previous findings using phosphorylation-mutant keratins (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The effect of okadaic acid and alkaline phosphatase on K8/K18 phosphorylation was confirmed by immunoblotting of the K8/K18 precipitates with anti-phospho-K8 and anti-phospho-K18 antibodies (Fig. 4 B). The VEVDA motif in the rod domain is found in K18 and K20, whereas the VEVDS motif is found in K19. Similar rod domain motifs, such as VEMDA, are present in other IF proteins including K14 (Fig. 1 B), whereas the DALD motif in the tail domain is unique to K18. Given the conserved nature of the rod domain motif (X1E/DX2DX3; X1-X3, aliphatic residues with caspase cleavage occurring at the aspartate between X2 and X3; Fig. 1 A) and the presence of K14 mutations at the aspartate of VEMD (D→G) and at X1 (V→M), X2 (M→R), and X3 (A→D) in patients with EBS, we asked whether these mutations have an effect on type I keratin fragmentation during apoptosis. To address this question, we generated several corresponding mutations in K14, K18, and K19 and tested the mutant constructs for susceptibility to caspase-mediated degradation in cell transfection systems. Mutation V236M in K18 or K19 to generate a WT K14-like caspase box (i.e. VEMD instead on VEVD) had no effect on caspase-mediated degradation of K18 (Fig.5 A) or K19 (Fig.5 C). Similarly, EBS-like mutations at the X1 or X2 positions of the caspase box of K14 (V270M or M272R, respectively) and of K19 (V234M or V236R, respectively) and at the X2 position of K18 (V236R) had no effect on keratin fragmentation upon apoptosis (Fig. 5; TableI). However, the X2 mutation of M (in K14) or V (in K18/19) to R generates an N-terminal fragment that migrates slightly faster on SDS-PAGE gels as compared with the equivalent N-terminal fragment generated with WT or other mutant K14, K18, or K19 fragments (Fig. 5, highlighted with anasterisk). It is unlikely that the altered migration is due to the valine to arginine substitution per se because the N-terminal fragment of K18 V220R migrates similarly to the WT K18 fragment (Fig. 5 B, compare lanes 1 and2). In addition, this faster-migrating N-terminal fragment does not appear to result from exposure of other potential cryptic caspase digestion sites (i.e. K18 Asp180 at177VEND or K18 Asp189 at 186KVID that may be exposed after the Val→Arg mutation in K18 V236R). For example, the double mutants K18 D180E/V236R or K18 D189E/V236R generate fragments with migration similar to that of K18 V236R (data not shown). Interestingly, an arginine substitution at the X1 position of K18 (V234R) blocks K18 cleavage at Asp237 (Fig.5 B, lane 3), thereby indicating that a basic residue substitution at the X1 caspase box position is likely to inhibit caspase enzyme-substrate recognition.Table IKeratin constructs and their degradation phenotypesType I keratinCaspase box sequenceConstruct mutationCleavage in rod domainK14270VEMD/ANone (wild-type)YesMEMD/AV270M (EBS)YesVERD/AM272R (EBS)Yes1-aAltered fragment migration in SDS-PAGE gels.VEMG/AD273G (EBS)NoVEME/AD273ENoVEMD/DA274D (EBS)YesK18234VEVD/ANone (wild-type)YesVEMD/AV236M (K14-like)YesVERD/AV236R (EBS-like)Yes1-aAltered fragment migration in SDS-PAGE gels.VEVG/AD237G (EBS-like)NoVEVE/AD237ENoVEVD/DA238D (EBS-like)YesREVD/AV234RNoK19234VEVD/SNone (wild-type)YesMEVD/SV234M (EBS-like)YesVEMD/SV236M (K14-like)YesVERD/SV236R (EBS-like)Yes1-aAltered fragment migration in SDS-PAGE gels.VEVE/SD237ENoVEVA/SD237ANoThe table summarizes the results obtained upon analysis of K14, K18, and K19 mutations within the conserved caspase box of the L1–2 region of the rod domain. Other details are provided in Figs. 2, 3, and 5. For some of the constructs, the results are stated in the table but not shown in the figures. Six constructs (one WT and five mutant) were analyzed for K14 and K19, and seven constructs (one WT and six mutant) were analyzed for K18. Mutant keratin cDNAs were generated as described under "Experimental Procedures" (mutant residue is shown in bold). Mutant or WT keratins were co-transfected with a WT type II keratin into BHK-21 cells, followed by SDS-PAGE separation and immunoblotting using a panel of keratin-specific antibodies to confirm the presence or absence of keratin cleavage (tabulated as "yes" and "no," respectively).1-a Altered fragment migration in SDS-PAGE gels. Open table in a new tab The table summarizes the results obtained upon analysis of K14, K18, and K19 mutations within the conserved caspase box of the L1–2 region of the rod domain. Other details are provided in Figs. 2, 3, and 5. For some of the constructs, the results are stated in the table but not shown in the figures. Six constructs (one WT and five mutant) were analyzed for K14 and K19, and seven constructs (one WT and six mutant) were analyzed for K18. Mutant keratin cDNAs were generated as described under "Experimental Procedures" (mutant residue is shown in bold). Mutant or WT keratins were co-transfected with a WT type II keratin into BHK-21 cells, followed by SDS-PAGE separation and immunoblotting using a panel of keratin-specific antibodies to confirm the presence or absence of keratin cleavage (tabulated as "yes" and "no," respectively). An EBS-like K14 VEMD→VEMG mutation and a similar K18 mutation (VEVD→VEVG) abolish caspase cleavage (Table I). As shown previously for K18 (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar) and shown here for K19 (D237E) and K14 (D273E), D→E mutations in these keratins also block caspase cleavage (Fig. 5,C and D). In addition, K18 VEVD→VEVE generates the 43-kDa fragment (K18 a+b in Fig. 2 A) due to caspase digestion at Asp396 in 393DALD, a site that is not found in other non-K18 type I keratins. Another EBS-like mutation at the X3 position in K14 (A274D) or K18 (A238D) does not affect susceptibility to caspase-mediated digestion (Table I), thereby indicating that the X3 position is insensitive to acidic charge perturbations. K18 (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar, 6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 7Leers M.P. Kolgen W. Bjorklund V. Bergman T. Tribbick G. Persson B. Bjorklund P. Ramaekers F.C. Bjorklund B. Nap M. Jornvall H. Schutte B. J. Pathol. 1999; 187: 567-572Crossref PubMed Scopus (573) Google Scholar) and K19 (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 8Prasad S. Soldatenkov V.A. Srinivasarao G. Dritschilo A. Int. J. Oncol. 1999; 14: 563-570PubMed Google Scholar) are the only keratins that have previously been demonstrated to undergo degradation during apoptosis. Degradation occurs primarily in type I keratins, with marked relative sparing of type II keratins as determined for K8, which is the only type II keratin studied in this context (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar, 6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Sparing of type II keratins may be related to differences of the context of the caspase box within the L1–2 region of the rod (Fig. 1 B, note the E→S substitution in type II keratins within the VEVD sequence), but type II keratins do posses other potential caspase recognition sequences (e.g. 77LEVD and 253LDMD in K8) that do not appear to be prominently cleaved. One important finding herein is that K14 is also a caspase substrate in transfected cells. This indicates that the remaining keratins of K12–17, in addition to desmin and neurofilament-L, are also likely to be caspase substrates because they all share the same VEMD motif, which differs slightly from the K18–20 (VEVD) motif. The type of keratin fragments generated during apoptosis may differ depending on the presence or absence of other caspase recognition motifs and their susceptibility to cleavage. In the case of K18, two well-defined cut sites occur as defined immunologically (7Leers M.P. Kolgen W. Bjorklund V. Bergman T. Tribbick G. Persson B. Bjorklund P. Ramaekers F.C. Bjorklund B. Nap M. Jornvall H. Schutte B. J. Pathol. 1999; 187: 567-572Crossref PubMed Scopus (573) Google Scholar) and molecularly (Fig. 2). These two sites undergo sequential caspase-mediated digestion (Fig. 6) with release of the small K18 tail fragment (397) from the K8/K18 complex, followed by cleavage at K18 Asp237 to generate two stable fragments (1–237 and 238–396) that remain associated with K8. This apoptosis-associated keratin cleavage is accompanied by significant reorganization of the keratin cytoskeletal network (5Caulin C. Salvesen G.S. Oshima R.G. J. Cell Biol. 1997; 138: 1379-1394Crossref PubMed Scopus (546) Google Scholar,20Liao J. Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 17565-17573Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Tinnemans M.M. Lenders M.H. ten Velde G.P. Ramaekers F.C. Schutte B. Eur. J. Cell Biol. 1995; 68: 35-46PubMed Google Scholar, 22van Engeland M. Kuijpers H.J. Ramaekers F.C. Reutelingsperger C.P. Schutte B. Exp. Cell Res. 1997; 235: 421-430Crossref PubMed Scopus (168) Google Scholar, 23MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (150) Google Scholar). Transient transfection of the K18, K19, and K14 mutants (with WT K8) did not have any significant effect on filament organization as determined by immunofluorescence staining (data not shown). Keratin hyperphosphorylation occurs as an early event upon exposure of cells to an apoptotic signal (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar,24Omary M.B. Ku N.O. Liao J. Price D. Subcell. Biochem. 1998; 31: 105-140PubMed Google Scholar). Mutation of the major K8 and K18 phosphorylation sites did not affect the susceptibility of caspase-mediated cleavage of K18 at Asp237, thereby indicating that dephosphorylation did not affect keratin degradation during apoptosis (6Ku N.O. Liao J. Omary M.B. J. Biol. Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). However, hyperphosphorylation does significantly inhibit caspase-3 in vitro digestibility of K18 at Asp237 (the second sequentially cut K18 site), but not at Asp396 (the first cut site) (Fig. 6). This raises the possibility that hyperphosphorylation of the remaining type I keratins, which are cleaved at the K18 Asp237-equivalent site (Fig.1 B), may also be protective. Several functional roles, acting alone or in concert, can be envisioned for keratin hyperphosphorylation during apoptosis: (i) a simple by-product of the apoptosis-associated activation of multiple kinases (e.g. Refs. 25Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita Ki K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1016) Google Scholar and 26Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar); if so, this favors a role for keratins as a phosphate reservoir or sink (24Omary M.B. Ku N.O. Liao J. Price D. Subcell. Biochem. 1998; 31: 105-140PubMed Google Scholar), (ii) a facilitator, alone or in concert with keratin degradation, of keratin filament reorganization during apoptosis, or (iii) a mechanism that either protects from apoptosis-induced damage (in this case degradation of keratins) or allows for a graded sequence of apoptotic events. The caspase box motif that is found within the L1–2 region of the rod domain of cytoplasmic IF proteins is a prototype caspase recognition motif, represented in type I keratins by X1E/DX2DX3 (with site of cleavage occurring at the D between X2 and X3; X1-X3, hydrophobic residues). Our results showed that replacement of X1 by an Arg prevents caspase-mediated degradation (Table I), which suggests that basic residue substitutions at X1 are likely to be incompatible with substrate-enzyme recognition. In contrast, the X2 or X3 positions were not affected by basic residue substitutions in that M/V→R mutations (i.e.VEMD/VEVD→VERD in K14, K18, or K19) or A→D mutations (VEMDA/VEVDA→VEMDD/VEVDDin K14 or K18), which mimic K14 mutations found in EBS patients, had no measurable effect on caspase-mediated cleavage of the type I keratin. These results, using an in vivo transfection system, are similar to what has been noted with in vitro peptide substrates in that the X2 position can tolerate a wide range of amino acids, whereas X1 seems to dictate caspase enzyme-type specificity (27Sleath P.R. Hendrickson R.C. Kronheim S.R. March C.J. Black R.A. J. Biol. Chem. 1990; 265: 14526-14528Abstract Full Text PDF PubMed Google Scholar, 28Talanian R.V. Quinlan C. Trautz S. 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Eur. J. Biochem. 1996; 241: 309-314Crossref PubMed Scopus (97) Google Scholar), respectively. However, the molecular mechanisms for generating tissue polypeptide antigen and tissue polypeptide-specific-like fragments are unknown, although caspase-mediated degradation and/or other protease activation are likely mechanisms. Hence, understanding the precise molecular changes that occur to keratins during apoptosis is an important first step in determining the significance of caspase-mediated keratin fragment formation and release in tumors. The presence of keratin mutations within the caspase box in patients with epidermal diseases raises the possibility that alterations in susceptibility to caspase-mediated cleavage could impact disease pathogenesis or alter susceptibility to other skin diseases. For example, apoptosis (and presumably subsequent keratin degradation) is a feature of several skin diseases and injury (e.g. Refs. 38Rosenthal D.S. Simbulan-Rosenthal C.M. Iyer S. Spoonde A. Smith W. Ray R. Smulson M.E. J. Invest. Dermatol. 1998; 111: 64-71Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholarand 39Gniadecki R. Jemec G.B. Thomsen B.M. Hansen M. Arch. Dermatol. Res. 1998; 290: 528-532Crossref PubMed Scopus (75) Google Scholar). Identification of the keratin cleavage sites during apoptosis will allow subsequent in vivo testing of the significance of mutations that inhibit keratin degradation on progression of apoptosis. Although keratin mutations are most common within the proximal region of the rod domain (12Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (836) Google Scholar, 13Irvine A.D. McLean W.H. Br. J. Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (329) Google Scholar), K14 mutations within the L1–2 domain have been described (14Rugg E.L. Morley S.M. Smith F.J. Boxer M. Tidman M.J. Navsaria H. Leigh I.M. Lane E.B. Nat. Genet. 1993; 5: 294-300Crossref PubMed Scopus (80) Google Scholar, 15Humphries M.M. Sheils D.M. Farrar G.J. Kumar-Singh R. Kenna P.F. Mansergh F.C. Jordan S.A. Young M. Humphries P. Hum. Mutat. 1993; 2: 37-42Crossref PubMed Scopus (53) Google Scholar, 16Chen H. Bonifas J.M. Matsumura K. Ikeda S. Leyden W.A. Epstein Jr., E.H. J. Invest. Dermatol. 1995; 105: 629-632Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 17Muller F.B. Kuster W. Bruckner-Tuderman L. Korge B.P. J. Invest. Dermatol. 1998; 111: 900-902Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Of the four such K14 mutations we tested, only the K14 D273G mutation prevented the caspase-mediated digestibility of K14 (Table I). Another K14 mutation (M272R) generated a K14 fragment with altered mobility on SDS-PAGE gels, and a similar mutation that was introduced in K18 and K19 also altered the migration of the resultant apoptotic fragment (Fig. 5). The cause of this altered migration is unclear, but it is unlikely to be due to the arginine mutationper se because introducing an arginine (V220R) proximally did not alter fragment migration (Fig. 5). Given that the type I keratins K12–14, K16, K17, and K18 have been associated with a variety of human diseases (12Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (836) Google Scholar, 13Irvine A.D. McLean W.H. Br. J. Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (329) Google Scholar), it is likely that additional mutations within L1–2 and the caspase box will be defined. If so, our results should facilitate the prediction of whether such mutations will interfere with caspase digestion. K15 and K17 were also recently shown biochemically to undergo apoptosis-induced digestion at the aspartate of the conserved VEMDA (40Badock V. Steinhusen U. Bommert K. Wittmann-Liebold B. Otto A. Cell Death Differ. 2001; 8: 308-315Crossref PubMed Scopus (27) Google Scholar). We are very grateful to Dr. Pierre Coulombe for providing us with cDNAs for wild-type K6, K14, and K19 and to Kris Morrow for preparing the figures.