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• W2014646858 abstract "Keratin 8 (K8) serine 73 occurs within a relatively conserved type II keratin motif (68NQSLLSPL) and becomes phosphorylated in cultured cells and organs during mitosis, cell stress, and apoptosis. Here we show that Ser-73 is exclusively phosphorylated in vitro by p38 mitogen-activated protein kinase. In cells, Ser-73 phosphorylation occurs in association with p38 kinase activation and is inhibited by SB203580 but not by PD98059. Transfection of K8 Ser-73 → Ala or K8 Ser-73 → Asp with K18 generates normal-appearing filaments. In contrast, exposure to okadaic acid results in keratin filament destabilization in cells expressing wild-type or Ser-73 → Asp K8, whereas Ser-73 → Ala K8-expressing cells maintain relatively stable filaments. p38 kinase associates with K8/18 immunoprecipitates and binds selectively with K8 using an in vitro overlay assay. Given that K1 Leu-160 → Pro (157NQSLLQPL →157NQSPLQPL) leads to epidermolytic hyperkeratosis, we tested and showed that the analogous K8 Leu-71 → Pro leads to K8 hyperphosphorylation by p38 kinase in vitro and in transfected cells, likely due to Ser-70 neo-phosphorylation, in association with significant keratin filament collapse upon cell exposure to okadaic acid. Hence, K8 Ser-73 is a physiologic phosphorylation site for p38 kinase, and its phosphorylation plays an important role in keratin filament reorganization. The Ser-73 → Ala-associated filament reorganization defect is rescued by a Ser-73 → Asp mutation. Also, disease-causing keratin mutations can modulate keratin phosphorylation and organization, which may affect disease pathogenesis. Keratin 8 (K8) serine 73 occurs within a relatively conserved type II keratin motif (68NQSLLSPL) and becomes phosphorylated in cultured cells and organs during mitosis, cell stress, and apoptosis. Here we show that Ser-73 is exclusively phosphorylated in vitro by p38 mitogen-activated protein kinase. In cells, Ser-73 phosphorylation occurs in association with p38 kinase activation and is inhibited by SB203580 but not by PD98059. Transfection of K8 Ser-73 → Ala or K8 Ser-73 → Asp with K18 generates normal-appearing filaments. In contrast, exposure to okadaic acid results in keratin filament destabilization in cells expressing wild-type or Ser-73 → Asp K8, whereas Ser-73 → Ala K8-expressing cells maintain relatively stable filaments. p38 kinase associates with K8/18 immunoprecipitates and binds selectively with K8 using an in vitro overlay assay. Given that K1 Leu-160 → Pro (157NQSLLQPL →157NQSPLQPL) leads to epidermolytic hyperkeratosis, we tested and showed that the analogous K8 Leu-71 → Pro leads to K8 hyperphosphorylation by p38 kinase in vitro and in transfected cells, likely due to Ser-70 neo-phosphorylation, in association with significant keratin filament collapse upon cell exposure to okadaic acid. Hence, K8 Ser-73 is a physiologic phosphorylation site for p38 kinase, and its phosphorylation plays an important role in keratin filament reorganization. The Ser-73 → Ala-associated filament reorganization defect is rescued by a Ser-73 → Asp mutation. Also, disease-causing keratin mutations can modulate keratin phosphorylation and organization, which may affect disease pathogenesis. The “soft” mucosal keratins (K) 1The abbreviations used are: KkeratinAbantibodyAnanisomycinEmpEmpigen BBIFintermediate filament(s)mAbmonoclonal antibodyMAPKmitogen-activated protein kinaseMMSmethyl methanesulfonateOAokadaic acidPBSphosphate-buffered salineSer(P)-phosphoserineSAPKstress-activated protein kinaseWTwild-typeBHKbaby hamster kidney 1The abbreviations used are: KkeratinAbantibodyAnanisomycinEmpEmpigen BBIFintermediate filament(s)mAbmonoclonal antibodyMAPKmitogen-activated protein kinaseMMSmethyl methanesulfonateOAokadaic acidPBSphosphate-buffered salineSer(P)-phosphoserineSAPKstress-activated protein kinaseWTwild-typeBHKbaby hamster kidney make up the intermediate filament (IF) proteins that are preferentially expressed in epithelial cells that line the inner and outer surfaces of animal tissues. These mucosal keratins consist of a large family (at least 20 members termed K1 to K20) of cytoplasmic proteins that are divided into relatively acidic type I (K9 to K20, pI < 6) and relatively basic type II (K1 to K8, pI ≥ 6) keratins (1.Calnek D. Quaroni A. Differentiation. 1993; 53: 95-104Crossref PubMed Scopus (61) Google Scholar, 2.Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4532) Google Scholar, 3.Moll R. Schiller D.L. Franke W.W. J. Cell Biol. 1990; 111: 567-580Crossref PubMed Scopus (335) Google Scholar, 4.Moll R. Zimbelmann R. Goldschmidt M.D. Keith M. Laufer J. Kasper M. Koch P.J. Franke W.W. Differentiation. 1993; 53: 75-93Crossref PubMed Scopus (187) Google Scholar). Epithelial cells generally express two or more keratin noncovalent heteropolymers in a 1:1 molar ratio of type I to II IFs, with an epithelial cell type-specific unique keratin complement. For example, single layered “simple type” epithelia express K8 and K18, with variable levels of K19 and K20 depending on the cell type, whereas keratinocytes express K5/14 or K1/10 basally and suprabasally, respectively. The prototype structure of all IF proteins, including keratins, consists of a central coiled-coil α-helix domain termed the “rod” that is flanked by non-α-helical N-terminal “head” and C-terminal “tail” domains (5.Herrmann H. Aebi U. Curr. Opin. Cell Biol. 2000; 12: 79-90Crossref PubMed Scopus (420) Google Scholar, 6.Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1281) Google Scholar). Notably, the head and tail domains of keratins contain most of the structural heterogeneity among IF proteins and also include the domains that undergo phosphorylation. This distribution correlation and other accumulating data (7.Eriksson J.E. Opal P. Goldman R.D. Curr. Opin. Cell Biol. 1992; 4: 99-104Crossref PubMed Scopus (126) Google Scholar, 8.Inagaki M. Matsuoka Y. Tsujimura K. Ando S. Tokui T. Takahashi T. Inagaki N. Bioessays. 1996; 18: 481-487Crossref Scopus (160) Google Scholar, 9.Ku N.O. Liao J. Chou C.F. Omary M.B. Cancer Metastasis Rev. 1996; 15: 429-444Crossref PubMed Scopus (100) Google Scholar, 10.Omary M.B. Ku N.O. Liao J. Price D. Subcell. Biochem. 1998; 31: 105-140PubMed Google Scholar, 11.Coulombe P.A. Omary M.B. Curr. Opin. Cell Biol. 2002; 14: 110-122Crossref PubMed Scopus (559) Google Scholar) strongly suggest that phosphorylation plays an important role in regulating the tissue-specific functional roles of the large keratin family. keratin antibody anisomycin Empigen BB intermediate filament(s) monoclonal antibody mitogen-activated protein kinase methyl methanesulfonate okadaic acid phosphate-buffered saline phosphoserine stress-activated protein kinase wild-type baby hamster kidney keratin antibody anisomycin Empigen BB intermediate filament(s) monoclonal antibody mitogen-activated protein kinase methyl methanesulfonate okadaic acid phosphate-buffered saline phosphoserine stress-activated protein kinase wild-type baby hamster kidney Although spectacular gains have been made in linking 14 of the more than 20 keratins to a number of skin, oral, esophageal, and liver diseases (12.Omary M.B. Ku N.O. Hepatology. 1997; 25: 1043-1048Crossref PubMed Scopus (89) Google Scholar, 13.Steinert P.M. Bale S.J. Trends Genet. 1993; 9: 280-284Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 14.Ku N.O. Gish R. Wright T.L. Omary M.B. N. Engl. J. Med. 2001; 344: 1580-1587Crossref PubMed Scopus (142) Google Scholar, 15.Irvine A.D. McLean W.H. Br. J. Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (329) Google Scholar, 16.Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (832) Google Scholar, 17.Fuchs E. Coulombe P.A. Cell. 1992; 69: 899-902Abstract Full Text PDF PubMed Scopus (122) Google Scholar), full appreciation of keratin and other IF protein function has been lagging. For some keratins, one clearly delineated function is to protect cells from mechanical and nonmechanical forms of injury, but how this occurs remains poorly understood (11.Coulombe P.A. Omary M.B. Curr. Opin. Cell Biol. 2002; 14: 110-122Crossref PubMed Scopus (559) Google Scholar, 12.Omary M.B. Ku N.O. Hepatology. 1997; 25: 1043-1048Crossref PubMed Scopus (89) Google Scholar, 18.Marceau N. Loranger A. Gilbert S. Daigle N. Champetier S. Biochem. Cell Biol. 2001; 79: 543-555Crossref PubMed Scopus (65) Google Scholar, 19.Omary M.B. Ku N.O. Toivola D.M. Hepatology. 2002; 35: 251-257Crossref PubMed Scopus (141) Google Scholar). Regardless, an intact keratin filament network and how keratin filaments are organized appear to be important effectors of this ability to maintain cellular integrity. This is borne out by many in vitro studies that correlated the importance of various keratin domains to form typical-appearing filaments and by the phenotypes that have been observed in patients with keratin diseases and in animal models that express different keratin mutants (11.Coulombe P.A. Omary M.B. Curr. Opin. Cell Biol. 2002; 14: 110-122Crossref PubMed Scopus (559) Google Scholar, 13.Steinert P.M. Bale S.J. Trends Genet. 1993; 9: 280-284Abstract Full Text PDF PubMed Scopus (49) Google Scholar,15.Irvine A.D. McLean W.H. Br. J. Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (329) Google Scholar, 16.Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (832) Google Scholar, 17.Fuchs E. Coulombe P.A. Cell. 1992; 69: 899-902Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 19.Omary M.B. Ku N.O. Toivola D.M. Hepatology. 2002; 35: 251-257Crossref PubMed Scopus (141) Google Scholar, 20.Magin T.M. Hesse M. Schroder R. Protoplasma. 2000; 211: 140-150Crossref Scopus (28) Google Scholar). Although perturbations within the highly conserved proximal and distal ends of the rod domain (which harbor most of the described disease-causing keratin mutations but lack any evidence of phosphorylation (10.Omary M.B. Ku N.O. Liao J. Price D. Subcell. Biochem. 1998; 31: 105-140PubMed Google Scholar, 15.Irvine A.D. McLean W.H. Br. J. Dermatol. 1999; 140: 815-828Crossref PubMed Scopus (329) Google Scholar)) have significant effects on filament organization in vivo and in vitro, keratin phosphorylation within the head and tail domains also plays a significant role in filament organization in vitro (8.Inagaki M. Matsuoka Y. Tsujimura K. Ando S. Tokui T. Takahashi T. Inagaki N. Bioessays. 1996; 18: 481-487Crossref Scopus (160) Google Scholar, 21.Yano T. Tokui T. Nishi Y. Nishizawa K. Shibata M. Kikuchi K. Tsuiki S. Yamauchi T. Inagaki M. Eur. J. Biochem. 1991; 197: 281-290Crossref PubMed Scopus (80) Google Scholar) and in vivo (9.Ku N.O. Liao J. Chou C.F. Omary M.B. Cancer Metastasis Rev. 1996; 15: 429-444Crossref PubMed Scopus (100) Google Scholar, 10.Omary M.B. Ku N.O. Liao J. Price D. Subcell. Biochem. 1998; 31: 105-140PubMed Google Scholar). In addition, keratin mutations within the head domains, which may modulate keratin phosphorylation, have been described. For example, mutations have been described that either introduce a new potential phosphorylation site (e.g.K1 157NQSLLQP →157NQSPLQP which renders Ser-159 a potential proline-directed kinase phosphorylation site (22.Chipev C.C. Korge B.P. Markova N. Bale S.J. DiGiovanna J.J. Compton J.G. Steinert P.M. Cell. 1992; 70: 821-828Abstract Full Text PDF PubMed Scopus (254) Google Scholar)) or remove possible phosphorylation sites (e.g. Ref. 23.Muller 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). Keratin phosphorylation has been most extensively studied in K8/18/19 (10.Omary M.B. Ku N.O. Liao J. Price D. Subcell. Biochem. 1998; 31: 105-140PubMed Google Scholar), due in part to the relative solubility of these keratins as compared with epidermal keratins (24.Chou C.F. Riopel C.L. Rott L.S. Omary M.B. J. Cell Sci. 1993; 105: 433-444Crossref PubMed Google Scholar). These studies resulted in the identification of several phosphorylation-mediated K8/18 functions. For example, K18 Ser-33 phosphorylation regulates keratin binding to the 14-3-3 family of proteins during mitosis, which in turn plays a role in keratin filament organization and solubility (25.Liao J. Omary M.B. J. Cell Biol. 1996; 133: 345-357Crossref PubMed Scopus (179) Google Scholar, 26.Ku N.O. Liao J. Omary M.B. EMBO J. 1998; 17: 1892-1906Crossref PubMed Scopus (194) Google Scholar). A direct role for keratin phosphorylation may also occur, as noted for K19, whereby mutation of its major phosphorylation site (Ser-35 → Ala) altered keratin filament organization in transiently transfected cells (27.Zhou X. Liao J. Hu L. Feng L. Omary M.B. J. Biol. Chem. 1999; 274: 12861-12866Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In addition, transgenic mouse studies showed that K18 Ser-52 phosphorylation facilitates a protective role against hepatotoxic injury (28.Ku N.O. Michie S.A. Soetikno R.M. Resurreccion E.Z. Broome R.L. Omary M.B. J. Cell Biol. 1998; 143: 2023-2032Crossref PubMed Scopus (86) Google Scholar), a finding that has provided direct evidence for a number of correlative data that document increased keratin phosphorylation in association with a variety of stresses in cultured cells and in intact animals (29.Ku N.O. Zhou X. Toivola D.M. Omary M.B. Am. J. Physiol. 1999; 277: G1108-G1137PubMed Google Scholar). In the case of human K8, three major in vivophosphorylation sites have been identified: Ser-23, Ser-431, and Ser-73. Ser-23 is a highly conserved site among all type II keratins, which suggests a common keratin function for this modification, whereas Ser-431 is a basally phosphorylated site that increases its phosphorylation specific activity during mitosis and upon exposure to epidermal growth factor in association with filament reorganization (30.Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 7556-7564Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In contrast K8 Ser-73 phosphorylation behaves like an on/off switch in cultured cells and in tissues, with phosphorylation being “on” during mitosis, a variety of cell stresses including heat and drug exposure, and during apoptosis (31.Liao J. Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 17565-17573Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Although the function of K8 Ser-73 phosphorylation was unknown, our hypothesis prior to embarking on this study was that its phosphorylation is likely to be important due to its on/off property and its association with important cell processes. Here we show that the mitogen-activated protein kinase (MAPK) p38 (reviewed in Refs.32.Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3641) Google Scholar, 33.Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2881) Google Scholar, 34.Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1390) Google Scholar, 35.Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3203) Google Scholar) is a physiologic kinase for K8 Ser-73 phosphorylation, and we demonstrate that K8 Ser-73 phosphorylation plays a significant role in keratin filament reorganization in response to the phosphatase inhibitor okadaic acid. Since K8 Ser-73 is proximal to a human disease mutation site in epidermal K1 (NQSLLQPL → NQSPLQPL, Ref. 22.Chipev C.C. Korge B.P. Markova N. Bale S.J. DiGiovanna J.J. Compton J.G. Steinert P.M. Cell. 1992; 70: 821-828Abstract Full Text PDF PubMed Scopus (254) Google Scholar; with K8 Ser-73 being part of the motif68NQSLLSPL of K8), we generated the equivalent K1 mutation in K8 (i.e. NQSLLSPL → NQSPLSPL) and showed that it increased K8 phosphorylation, as compared with wild-type K8. This skin disease-causing mutation also resulted in significant keratin filament collapse in the presence of okadaic acid. Therefore, K8 Ser-73 phosphorylation plays an important role in modulating keratin filament reorganization. In addition, this is the first demonstration that human keratin disease-causing mutations can indeed result in keratin hyperphosphorylation and that such hyperphosphorylation can affect keratin filament organization, which in turn may contribute to disease pathogenesis. The antibodies (Ab) used are as follows: L2A1 mouse monoclonal antibody (mAb) that recognizes human K18 (24.Chou C.F. Riopel C.L. Rott L.S. Omary M.B. J. Cell Sci. 1993; 105: 433-444Crossref PubMed Google Scholar); mAb LJ4 that recognizes K8 Ser(P)-73 (31.Liao J. Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 17565-17573Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar); mAb 5B3 that recognizes K8 Ser(P)-431 (30.Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 7556-7564Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar); rabbit Ab 8250 that recognizes K18 Ser(P)-33 (26.Ku N.O. Liao J. Omary M.B. EMBO J. 1998; 17: 1892-1906Crossref PubMed Scopus (194) Google Scholar); rabbit Ab 3055 that recognizes K18 Ser(P)-52 (36.Liao J. Lowthert L.A. Ku N.O. Fernandez R. Omary M.B. J. Cell Biol. 1995; 131: 1291-1301Crossref PubMed Scopus (73) Google Scholar); mAb M20 (NeoMarkers; Freemont, CA) that recognizes K8; and anti-FLAG antibody (Sigma). Other reagents used are as follows: anisomycin (An); Empigen BB (Emp); p42 kinase; c-Jun N-terminal kinase (JNK); p38 kinase (Calbiochem-Novabiochem); methyl methanesulfonate (MMS) (Aldrich); PD98059, anti-p38, and anti-phospho-p38 antibodies (New England Biolabs, Beverly, MA); SB203580 (kindly provided by Dr. John Lee, SmithKline Beecham Pharmaceuticals, King of Prussia, PA); orthophosphate (32PO4); and [γ-32P]ATP (PerkinElmer Life Sciences). HT-29 (human colon), BHK (hamster kidney), and NIH-3T3 (mouse fibroblast) cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured as recommended by the supplier. To activate p38 kinase, cells were incubated with An (10 μg/ml, 0–20 h) or with MMS (0.1 or 1 mg/ml, 0–24 h). Cells were then solubilized with 2% SDS-containing sample buffer (37.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207139) Google Scholar) followed by shearing of the DNA with a 27-gauge needle and then boiling for 2 min to generate a total cell lysate. Alternatively, cells were processed for immunoprecipitation as described below. For the kinase inhibitors SB203580 (p38 kinase) and PD98059 (MAPK kinase), cells were preincubated with these compounds (20 and 100 μm, respectively) for 1 h and then treated with An for 2 h. Transiently transfected cells were grown on coverslips and fixed 3 days after transfection, using 100% methanol (−20 °C) for 3 min. Staining was done as described (26.Ku N.O. Liao J. Omary M.B. EMBO J. 1998; 17: 1892-1906Crossref PubMed Scopus (194) Google Scholar). For okadaic acid (OA) treatment, OA (1 μg/ml) was added to the transfected cells for 2 h before fixation and processing. Fluorescence was analyzed using a Bio-Rad MRC1024 confocal laser scanning and a Nikon TE300 inverted microscope. Cells co-transfected with WT K18 and one of the four K8 constructs (WT, S73A, S73D, or L71P) were scored, after treatment with OA, based on their filament organization as follows: (i) cells with residual filaments, (ii) cells with fine dots but without any residual filaments, and (iii) cells with large dots. The K8 mutants K8 Ser-73 → Ala (S73A), S73D, and L71P were generated using a TransformerTM mutagenesis kit (CLONTECH Laboratories Inc., Palo Alto, CA) as recommended by the supplier. Wild-type (WT) K8, WT K18, or mutant K8 cDNAs were subcloned into the pMRB101 mammalian expression vector under control of the hCMV promoter. The FLAG-tagged α-isoform of WT p38 or p38 AF (kinase-inactive form due to double mutation at the phosphorylation sites, T180A and Y182F; Ref. 38.Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2412) Google Scholar) were used to overexpress the p38 proteins in BHK cells with keratin constructs. Transient transfections into NIH-3T3 or BHK cells were done using LipofectAMINE as recommended by the supplier. The NIH-3T3 cells were used for immunofluorescence experiments because they provided a well formed keratin filament-staining pattern, whereas BHK cells were used to generate keratins for the biochemical experiments since they had a higher transfection efficiency. Immunoprecipitation was carried out by solubilizing cells with 1% Emp (1 h, 4 °C) in buffer A (phosphate-buffered saline (PBS) (pH 7.4) containing 5 mmEDTA, 0.1 mm phenylmethanesulfonyl fluoride, 10 μm pepstatin, 10 μm leupeptin, 25 μg/ml aprotinin, and 1 μg/ml OA) or by solubilizing cells with 1% Nonidet P-40 in buffer A. After pelleting (15 min; 16,000 ×g), keratins were immunoprecipitated from the supernatant using Sepharose-conjugated L2A1 followed by washing, analysis by SDS-PAGE (37.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207139) Google Scholar), and then staining with Coomassie Blue. For immunoblotting, gels were transferred to membranes followed by blotting (39.Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44912) Google Scholar) with individual anti-keratin antibodies. Bound antibodies were visualized with peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Two-dimensional chymotryptic phosphopeptide mapping was carried out exactly as described (30.Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 7556-7564Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 40.Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1275) Google Scholar) using electrophoresis in the first (horizontal) dimension and chromatography in the second (vertical) dimension. The overlay assay was performed as described (41.Liao J. Lowthert L.A. Ghori N. Omary M.B. J. Biol. Chem. 1995; 270: 915-922Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) with minor modifications. Briefly, total lysate and K8/18 immunoprecipitates from HT-29 cells were analyzed using SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane (4 °C). The membrane was blocked with 3% BSA in PBS for 2 days, followed by incubation with 1 μg/ml p38 kinase in PBS with 0.05% Tween and 0.1% BSA for 2 h (22 °C). After washing, the membrane was incubated with anti-p38 antibody for immunoblotting. In vitro kinase reactions were carried out using K8/18 immunoprecipitates. For each of the kinases used (p38, p42, and Jun kinases), the buffers provided by the supplier were used as recommended. Immunoprecipitates of K8/18 were washed two times with the respective kinase buffer (in addition to the routine washings as part of immunoprecipitation) and then incubated with 5 μCi of [γ-32P]ATP, the kinase, and 20 μm ATP (10 min in a total volume of 25 μl). The kinase reaction was quenched by adding 4 times the normal concentration of Laemmli sample buffer, followed by boiling for 90 s and then analysis by SDS-PAGE and autoradiography. Metabolic labeling with [32P]orthophosphate was done by incubating cells (in 100-mm dishes) with 5 ml of phosphate-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum and 100 mm glutamine for 30 min followed by the addition of 50 μl of normal medium and 250 μCi/ml [32P]orthophosphate. After labeling for 5 h, keratins were immunoprecipitated from the detergent-solubilized cells using mAb L2A1 and then analyzed by preparative SDS-PAGE and Coomassie staining, followed by isolation of the individual keratin-stained bands for peptide mapping. We identified previously (31.Liao J. Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 17565-17573Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) K8 Ser-73 as a K8 phosphorylation site using what we termed a “reverse immunologic” approach. This was aided by an antibody termed LJ4, which was generated by immunizing mice with keratins that were purified from okadaic acid-treated HT-29 cells. As shown previously (31.Liao J. Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 17565-17573Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and exemplified in Fig. 1A, mAb LJ4 selectively recognizes the hyperphosphorylated and slightly slower migrating K8 species termed HK8. The HK8 species are present in very small amounts in exponentially growing HT-29 cells as determined by immunoprecipitation with mAb L2A1, which recognizes the entire keratin pool (31.Liao J. Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 17565-17573Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), but become markedly enriched after immunoprecipitation with mAb LJ4. The LJ4 Ab recognizes HK8 exclusively (Fig. 1B, lane 1), and its reactivity is abolished if Ser-73 is mutated to an alanine (S73A) (Fig. 1B, lane 2). However, LJ4 does recognize K8 S73D weakly (Fig. 1B, lane 3), which migrates slightly faster than HK8 and a bit slower than K8, such that LJ4 has almost equal binding intensity to the barely visible Coomassie-stained HK8 as compared with the strongly staining K8 S73D species (Fig. 1B). We compared the in vitro phosphorylation of K8 by the proline-directed MAPKs p38 and p42, given the sequence context of K8 Ser-73 (71LLSPL) and the previous observation (31.Liao J. Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 17565-17573Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) that K8 Ser-73 becomes phosphorylated during heat stress and apoptosis. As shown in Fig. 1C, p38 kinase generates the radiolabeled HK8 species exclusively (a signature of Ser-73 phosphorylation), whereas p42 kinase generates phosphorylated K8 and HK8 (K8 > HK8; compare lanes 2 and 3). Mutation of the two major K8 phosphorylation sites, Ser-23 and Ser-431 (30.Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 7556-7564Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), did not affect formation of HK8 upon in vitro phosphorylation of K8/18 precipitates with p38 kinase (Fig. 2A, lanes 2 and 4). In contrast, mutation of K8 Ser-73 abolished formation of the HK8 species and resulted in barely detectable K8 phosphorylation (Fig. 2A, lane 3) that is likely due to Ser-431 phosphorylation (the only other K8 potential proline-directed kinase site, with the sequence 429LTSPG). The specificity of p38 kinase toward K8 Ser-73 is evident by the minimal formation of HK8 by p42 kinase (Fig. 2B) and the nearly equal generation of phospho-K8 and HK8 species by JNK (Fig. 2C). K8 Ser-23, which is a major basally phosphorylated K8 site (30.Ku N.O. Omary M.B. J. Biol. Chem. 1997; 272: 7556-7564Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), is not phosphorylated in vitro by any of the three tested MAPKs, whereas K8 Ser-431 phosphorylation occurs by p42 and JNK but not by p38 kinase (Fig. 2). Hence, the in vitro kinase assays of WT and mutant K8 immunoprecipitates indicate that both JNK and p42 phosphorylate K8 Ser-431 and Ser-73 relatively promiscuously, albeit to varied levels, in marked contrast to the selectivity of p38 kinase to the K8 Ser-73 site. In addition, phosphorylation of K8 Ser-73 does not appear to impact on K8 Ser-431 phosphorylation and vice versa. Given the findings in Figs. 1 and 2, we explored the role of p38 kinase as a potential in vivo K8 kinase by utilizing known specific activators and inhibitors of p38 kinase and by comparing phosphopeptide maps of in vivo versus in vitro p38-phosphorylated K8. As shown in Fig. 3A, activation of p38 kinase in cultured HT-29 cells by An (42.Cano E. Doza Y.N. Ben-Levy R. Cohen P. Mahadevan L.C. Oncogene. 1996; 12: 805-812PubMed Google Scholar), as determined by p38 phosphorylation, is associated with rapid K8 Ser-73 phosphorylation. Similarly, the alkylating agent MMS, a known p38 kinase and JNK activator (43.Wilhelm D. Bender K. Knebel A. Angel P. Mol. Cell. Biol. 1997; 17: 4792-4800Crossref PubMed Scopus (225) Google Scholar), generates the HK8 species in a dose- and time-dependent fashion (Fig. 3B). Inhibition of An-induced p38 kinase activation with the specific inhibitor compound SB203580 abrogated K8 Ser-73 phosphorylation (Fig. 3C). In contrast, inhibition of ERK1/2 kinase activation with compound PD98059 did not significantly affect K8 Ser-73 phosphorylation but did inhibit K8 Ser-431 phosphorylation as determined by blotting with mAb 5B3 (Fig. 3D). A comparison of the chymotryptic phosphopeptide maps of K8 and HK8 that are isolated from in vivo phosphorylated cells shows that HK8 differs from K8 by the presence of peptides 2–5 and by the absence of the peptide highlighted by an unnumbered arrow (Fig. 4, a and b). Interestingly, the phosphopeptide profile of HK8 that is generated by in vitro phosphorylation of K8 with p38 kinase shows five major peptides (Fig. 4c) that co-migrate with peptides 1–5 that are isolated from in vivo labeled HK8. This is confirmed by mixing in vitro and in vivo labeled K8 (Fig. 4d) and by mixing in vivo labeled HK8 with p38-labeled K8 (not shown). The five peptides are generated by incomplete chymotryptic digestion (not shown). Taken together, these results suggest that a p38-like kinase is likely to be involved, in vivo, in K8 phosphorylation at Ser-73. We further substantiated in vivo p38 phosphorylation of K8 Ser-73 by comparing K8 Ser-73 phosphorylation in BHK cells transfected with FLAG-tagged human WT p38 or kinase-inactive p38 AF (Fig. 5A). The overexpressed p38α proteins are detected with anti-FLAG and anti-human p38 antibodies. As anticipated, p38 AF is not recognized by phospho-p38 antibody, and K8 Ser-73 phosphorylation increases in BHK cells that overexpress WT but not AF p38 (Fig. 5A, lanes 1–3). In addition, WT and AF p38 kinases co-immunoprecipitate with K8/18 in tr" @default.
• W2014646858 created "2016-06-24" @default.
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• W2014646858 title "Keratin 8 Phosphorylation by p38 Kinase Regulates Cellular Keratin Filament Reorganization" @default.
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