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`Editor-in-Chief
`
`Norton B. Gilula
`Department of Cell Biology, MB6, The Scripps Research Institute, 10550 North
`Torrey Pines Rd., La Jolla, CA 92037. Tel.: (619) 784-9880. Fax: (619) 784-2345.
`E-mail: thejcb@scripps.edu
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`Associate Editors
`Don W. Cleveland
`Ludwig Institute for Cancer Research, University of California at San Diego,
`Room 3080 CMM-East, Mailstop 0660, 9500 Gilman Drive, La Jolla, CA 92093.
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`Werner Franke
`Division of Cell Biology, German Cancer Research Center, Im Neuenheimer
`Feld 280,69120 Heidelberg, F.R.G. Tel.: (49) 6221 42 3451.
`Fax: (49) 6221 42 3452. E-mail: jcb@dkfz-heidelberg.de
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`Ira Mellman
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`Tl1e Journal of
`
`Volume 143, Number 7,
`December 28, 1998
`
`W. James Nelson
`Department of Molecular and Cellular Physiology, Beckman Center B 121,
`Stanford University School of Medicine, Stanford. CA 94305. Tel.: (650)
`725-7596. Fax: (650) 498-5286. E-mail: wjnelson@leland.stanford.edu
`Louis F. Reichardt
`Neuroscience Program, Howard Hughes Medical Institute, Departments of
`Physiology and Biochemistry & Biophysics, University of California, Rm. U426,
`San Francisco, CA 94143. Tel.: (415) 476-3976. Fax: (415) 476-9914. E-mail:
`lfr@cgl.ucsf.edu
`Randy Schekman
`Department of Molecular and Cell Biology, University of California, 401 Barker
`Hall, Berkeley, CA 94720. Tel.: (510) 643-8358. Fax: (510) 643-8493.
`E-mail: schekman@uclink4.berkeley.edu
`Kenneth M. Yamada
`Craniofacial Developmental Biology and Regeneration Branch, National
`Institute of Dental Research, National Institutes of Health, Building 30, Room
`421, 30 Convent Drive MSC 4370, Bethesda, MD 20892-4370. Fax: (301) 365-2656.
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`Contents:
`The Journal of Cell Biology
`Volume 143, Number 7, December 28, 1998
`
`In Brief
`Movement by cable or capture. Gathering
`together unfolded proteins. Not all sarcoglycans
`are equal. Nuclear pore complexes and spindle
`pole bodies share a component. Early functions
`for desmosomes. A sex -specific homeodomain
`protein in algae.
`W.A. Wells
`
`Regular Articles ·
`INCENP centromere and spindle targeting:
`Identification of essential conserved motifs and
`involvement of heterochromatin protein HP1.
`A.M. Ainsztein, S.E. Kandels-Lewis, A.M. Mackay,
`and W.C. Earnshaw
`
`1763
`
`177 5 Fission yeast Bub 1 is a mitotic centromere
`protein essential for the spindle checkpoint and
`the preservation of correct ploidy through
`mitosis.
`P. Bernard, K. Hardwick, and J.-P. Javerzat
`
`1789 Saccharomyces cerevisiae Ndclp is a shared
`component of nuclear pore complexes and
`spindle pole bodies.
`H.J. Chial, M.P. Rout, T.H. Giddings, Jr.,
`and M. Winey
`
`1801 Functional analysis of Tpr: Identification of
`nuclear pore complex association and nuclear
`localization domains and a role in mRNA
`export.
`P. Bangs, B. Burke, C. Powers, R. Craig, A. Purohit,
`and S. Doxsey
`
`1813 Specific binding of the karyopherin Kap121p to
`a subunit of the nuclear pore complex containing
`Nup53p, Nup59p, and Nup170p.
`M. Marelli, J.D. Aitchison, and R.W. Wozniak
`
`1831 Homotypic fusion of immature secretory
`granules during maturation in a cell-free assay.
`S. Urbe, L.J. Page, and S.A. Tooze
`
`1845 Biochemical and functional studies of cortical
`vesicle fusion: The SNARE complex and Ca2+
`sensitivity.
`J.R. Coorssen, P.S. Blank, M. Tahara,
`and J. Zimmerberg
`
`1859 Pex18p and Pex21p, a novel pair of related
`peroxins essential for peroxisomal targeting by
`the PTS2 pathway.
`P.E. Purdue, X. Yang, and P.B. Lazarow
`
`1871 Redundant and distinct functions for dynamin-1
`and dynamin-2 isoforms.
`Y. Altschuler, S.M. Barbas, L.J. Terlecky, K. Tang,
`S. Hardy, K.E. Mostov, and S.L. Schmid
`
`1883 Aggresomes: A cellular response to misfolded
`proteins.
`J.A. Johnston, C.L. Ward, and R.R. Kopito
`
`1899 Visualization of melanosome dynamics within
`wild-type and dilute melanocytes suggests a
`paradigm for myosin V function in vivo.
`X. Wu, B. Bowers, K. Rao, Q. Wei,
`and J.A. Hammer III
`
`1919 Visualization and molecular analysis of actin
`assembly in living cells.
`D.A. Schafer, M.D. Welch, L.M. Machesky,
`P.C. Bridgman, S.M. Meyer, and J.A. Cooper
`
`1931 Tropomyosin-containing actin cables direct the
`Myo2p-dependent polarized delivery of secretory
`vesicles in budding yeast.
`D.W. Pruyne, D.H. Schott, and A. Bretscher
`
`194 7 Dual function of Cyk2, a cdc15/PSTPIP family
`protein, in regulating actomyosin ring dynamics
`and septin distribution.
`J. Lippincott and R. Li
`
`1961 Nonuniform microtubular polarity established
`by CH01/MKLP1 motor protein is necessary for
`process formation of podocytes.
`N. Kobayashi, J. Reiser, W. Kriz, R. Kuriyama,
`and P. Mundel
`
`1971 A gamete-specific, sex-limited homeodomain
`protein in Chlamydomonas.
`V. Kurvari, N.V. Grishin, and W.J. Snell
`
`Contents continued
`
`Cover picture: An ES .0 day mouse embryo was labeled with E-cadherin
`(red), desmoplakin (green), and DAPI (blue) and viewed using confocal
`microscopy. Adherens junctions are located between cells of the extraem(cid:173)
`bryonic tissues (ectoplacental cone and visceral endoderm) and embry(cid:173)
`onic tissue (primitive ectodermal), while desmosomes at this stage are lo(cid:173)
`cated only between cells in the extraembryonic tissues. See related article
`in this issue by Gallicano et al., 2009-2022.
`
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`1981 ARFl 1nediates paxillin recruitment to focal
`adhesions and potentiates Rho-stimulated stress
`fiber formation in intact and permeabilized Swiss
`3T3 fibroblasts.
`J.C. Norman, D. Jones, S.T. Barry, M.R. Holt,
`S. Cockcroft, and D.R. Critchley
`
`1997
`
`Regulation of the cell cycle by focal adhesion
`kinase.
`J.-H. Zhao, H. Reiske, and J.-L. Guan
`
`2009 Desmoplakin is required early in development
`for assembly of· desmosomes and cytoskeletal
`linkage.
`G.I. Gallicano, P. Kouklis, C. Bauer, M. Yin,
`V. Vasioukhin, L. Degenstein, and E. Fuchs
`
`2023 Mutation of a major. keratin phosphorylation site
`predisposes to hepatotoxic injury in transgenic
`mice.
`N.-0. Ku, S.A. Michie, R.M. Soetikno,
`E.Z. Resurreccion, R.L. Broome, and M.B. Omary
`
`2033 Molecular organization of sarcoglycan complex
`in mouse myotubes in culture.
`Y.-m. Chan, C.G. Bonnemann, H.G.W. Lidov,
`and L.M. Kunkel
`
`2045 Functional characteristics of ES cell-derived
`cardiac precursor cells identified by
`tissue-specific expression of the green
`fluorescent protein.
`E. Kolossov, B.K. Fleischmann, Q. Liu, W. Bloch,
`S. Viatchenko-Karpinski, 0. Manzke, G.J. Ji,
`H. Bohlen, K. Addicks, and J. Hescheler
`
`2057 Autocrine tumor necrosis factor (TNF) and
`lymphotoxin (L T) a differentially modulate
`cellular sensitivity to TNF/LT-a cytotoxicity in
`L929 cells.
`E. Decoster, S. Cornelis, B. Vanhaesebroeck,
`and W. Fiers
`
`2067 The cell adhesion molecule L1 is
`developmentally regulated in the renal
`epithelium and is involved in kidney branching
`morphogenesis.
`H. Debiec, E.I. Christensen, and P.M. Ronco
`2081 Activation of avl33 on vascular cells controls
`recognition of prothrombin.
`T.V. Byzova and E.F. Plow
`
`2093 ADDITIONS AND CORRECTIONS
`2095 AUTHOR INDEX FOR VOLUMES 140-143
`2131 SUBJECT INDEX FOR VOLUMES 140-143
`2157 ACKNOWLEDGMENT
`
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`Mutation of a Major Keratin Phosphorylation Site
`Predisposes to Hepatotoxic Injury in Transgenic Mice
`Nam-On Ku,*ll Sara A. Michie,+ll Roy M. Soetikno,*ll Evelyn Z. Resurreccion,+ll Rosemary L. Broome,§ll
`and M. Bishr Omary*ll
`*Department of Medicine, *Department of Pathology, and §Department of Veterinary Medicine, Veterans Administration Palo
`Alto Health Care System, Palo Alto, CA 94304; and the IIDigestive Disease Center, Stanford University School of Medicine,
`Palo Alto, California 94305
`
`Abstract. Simple epithelia express keratins 8 (K8) and
`18 (K18) as their major intermediate filament (IF) pro(cid:173)
`teins. One important physjologic function of K8/18 is to
`protect hepatocytes from drug-induced liver injury. Al(cid:173)
`though the mechanism of this protection is unknown,
`marked K8/18 hyperphosphorylation occurs in associa(cid:173)
`tion with a variety of cell stresses and during mitosis.
`This increase in keratin phosphorylation involves mul(cid:173)
`tiple sites including human K18 serine-( ser )52, which is
`a major K18 phosphorylation site. We studied the sig(cid:173)
`nificance of keratin hyperphosphorylation and focused
`on K18 ser52 by generating transgenic mice that over(cid:173)
`express a human genomic K18 ser52-?ala mutant
`(S52A) and compared them with mice that overexpress,
`at similar levels, wild-type (WT) human K18. Abroga(cid:173)
`tion of K18 ser52 phosphorylation did not affect fila(cid:173)
`ment organization after partial hepatectomy nor the
`
`ability of mouse livers to regenerate. However, expo(cid:173)
`sure of S52A-expressing mice to the hepatotoxins,
`griseofulvin or microcystin, which are associated with
`K18 ser52 and other keratin phosphorylation changes,
`resulted in more dramatic hepatotoxicity as compared
`with WT K18-expressing mice. Our results demonstrate
`that K18 ser52 phosphorylation plays a physiologic role
`in protecting hepatocytes from stress-induced liver in(cid:173)
`jury. Since hepatotoxins are associated with increased
`keratin phosphorylation at multiple sites, it is likely that
`unique sites aside from K18 ser52, and phosphorylation
`sites on other IF proteins, also participate in protection
`from cell stress.
`
`Key words: keratins • phosphorylation • intermediate
`filaments • transgenic mice • liver
`
`I NTERMEDIATE filament (IF)1 proteins are one of the
`
`three major cytoskeletal protein groups, that also in(cid:173)
`clude microfilaments and microtubules (reviewed in
`reference 14, 34, 49). A comparison of the three major cy(cid:173)
`toskeletal protein groups shows several distinguishing
`unique features of IF proteins: they have nuclear (i.e., the
`lamins) and many cytoplasmic members, their cytoplasmic
`members appear to be expressed only in higher eucaryotes
`in a tissue preferential manner, and mutations of IF pro(cid:173)
`teins cause a variety of human diseases (15). Among the
`diverse IF protein family, the keratin subgroup is the larg-
`
`Address correspondence to Bishr Omary, Palo Alto VA Medical Center,
`3801 Miranda Avenue, 154J, Palo Alto, CA 94304. Fax: (415) 852-3259.
`Address reprint requests to Nam-On Ku, Palo Alto VA Medical Cen(cid:173)
`ter, 3801 Miranda Avenue, 154J, Palo Alto, CA 94304. Fax: (415) 852-
`3259.
`
`1. Abbreviations used in this paper: Emp, Empigen BB; GF, griseofulvin;
`IF, intermediate filament(s); K, keratin; MLR, microcystin LR; pS, phos(cid:173)
`pho-serine; PVDF, polyvinylidene difluoride; S52A, transgenic mice that
`overexpress ser52--7ala K18; TG2, transgenic mice that overexpress wild(cid:173)
`type human K18; WT, wild-type.
`
`est and is specifically expressed in epithelial cells ( 44).
`Aside from the "hard" keratins that are found in epider(cid:173)
`mal appendages (e.g., hair and nails), the "soft" keratins
`(K) consist of >20 members that are divided into rela(cid:173)
`tively basic type II (K1-K8) and acidic type I (K9-K20)
`keratins ( 44). All epithelial cells express at least one type I
`and one type II keratin that associate, noncovalently, to
`form extended filamentous arrays. In general, epithelial
`cells express a dominant unique keratin pair depending on
`the epithelial cell type. For example, glandular epithelia
`express K8 and K18 (with variable levels of K19 and K20),
`whereas keratinocytes preferentially express K5/14 basally
`and Kl/10 suprabasally. Although keratin function(s) re(cid:173)
`main poorly understood, one clear function is to provide
`mechanical integrity to cells including those in epidermis
`(15), cornea (19) and liver ( 46). This was clearly demon(cid:173)
`strated by the phenotypes of several human diseases that
`are caused by keratin mutations and by several transgenic
`animal models that express mutant keratins (15, 43, 46).
`All IF proteins have a prototype structure consisting of
`a central coiled-coil a.-helix (310-350 amino acids) that is
`surrounded by globular NHz-terminal head and COOH-
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`The Journal of Cell Biology, Volume 143, Number 7, December 28, 1998 2023-2032
`http://www.jcb.org
`2023
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`terminal tail domains of variable length (14, 34, 49). The
`globular end domains provide most of the structural het(cid:173)
`erogeneity among IF proteins and contain all currently
`known phosphorylation and glycosylation sites, in contrast
`with the nonmodified rod domain (reviewed in 18, 26, 47).
`The exclusive presence of keratin phosphorylation within
`the structurally heterogeneous head and tail domains sug(cid:173)
`gests that this modification may play a regulatory role in
`the presumed tissue-specific function of these proteins. In
`the case of K18, ser52 is its major phosphorylation site
`during interphase. K18 ser52 phosphorylation is highly dy(cid:173)
`namic, increases two to three times during mitosis, and ap(cid:173)
`pears to be important for filament reorganization (23, 37).
`A potential role for K18 ser52 in filament organization
`was implicated based on transfection of a K18 ser52--7ala
`mutant, which results in a keratin filamentous array that is
`twofold less able to reorganize its filaments, after treat(cid:173)
`ment with colcemid or okadaic acid, as compared with WT
`K18-transfected cells (23). Several other K8 and Kl8 phos(cid:173)
`phorylation sites have been identified including K18 ser33
`that regulates binding to 14-3-3 proteins (30), K8 ser23
`that is highly conserved among all type II keratins (24), K8
`ser431 that is phosphorylated by MAP kinase upon EGF
`stimulation (24), and K8 ser73 that becomes phosphory(cid:173)
`lated during mitosis, cell stress, and apoptosis ( 40).
`Two observations led us to investigate keratin phosphor(cid:173)
`ylation in transgenic mice in situ. First, disruption of K8/18
`filaments in transgenic mice after expression of a domi(cid:173)
`nant negative K18 arg89--7cys results in hepatocyte keratin
`IF collapse in association with chronic hepatitis (25) and
`increased susceptibility to drug-induced liver injury (27).
`This, coupled with the abnormal liver phenotype of K8-
`null mice (4, 5, 41) and mice that express K14 ectopically in
`the liver (3) indicated that K8/18 play an important role in
`helping hepatocytes cope with cell stress. Such a functional
`role for keratin was not discernible before the use of trans(cid:173)
`genic animal models as was done first for K14 (54) and
`subsequently several other IF proteins (15), and the initial
`identification of keratin mutations in the human blistering
`skin disease epidermolysis bullosa simplex (6, 13, 33). Sec(cid:173)
`ond, a variety of stress conditions in cultured cells and
`intact animals are associated with significant K8/18 hy(cid:173)
`perphosphorylation, including rotavirus infection or heat
`stress in cultured cells (38), apoptosis (8, 28), and drug(cid:173)
`induced liver injury using the anti-fungal agent griseoful(cid:173)
`vin (27) or the phosphatase inhibitor microcystin LR (MLR;
`45, 51, 52). Given that ser52 is a major K18 phosphoryla(cid:173)
`tion site, we sought to determine its physiologic function
`by generating transgenic mice that overexpress a K18
`ser52--7ala mutant and comparing the phenotype and ker(cid:173)
`atin phosphorylation in these mice with the well described
`transgenic mice that overexpress WT human K18 (termed
`TG2; references 1, 25, 27). This represents the first report
`that we are aware of that utilizes transgenic animals to
`study the significance of a single phosphorylation site of a
`protein.
`
`Materials and Methods
`
`Cell Culture, Antibodies, and Reagents
`NIH-3T3 (mouse fibroblast) and HT29 (human colon) cells (American
`
`Type Culture Collection, Manassas, VA) were cultured as recommended
`by the supplier. Antibodies (Ab) used were: L2A1 mouse mAb that rec(cid:173)
`ognizes human K18 without cross-reacting with mouse keratins (11, 25);
`Troma I rat mAb that recognizes mouse K8 (Developmental Studies Hy(cid:173)
`bridoma Bank, University of Iowa, Iowa City, IA); rabbit Ab 8592 that
`was raised against human K8/18 (25); rabbit Ab 3055 that recognizes
`phospho-ser52 (pS52) of human K18 and does not cross-react with the
`corresponding phosphorylation site in mouse K18 that has a different anti(cid:173)
`genic context (37); rabbit Ab 8250 that recognizes human K18 pS33 and
`does cross-react with the equivalent mouse phosphorylation site (30);
`mAb LJ4 that recognizes K8 pS73 ( 40); anti-mouse K18 (provided by
`Robert Oshima); and anti-plectin and anti-desmoplakin Abs (provided by
`Harald Herrmann and Manijeh Pasdar, respectively). Other reagents used
`were: microcystin-LR (Alexis Corp., San Diego, CA), griseofulvin (GF;
`Sigma Chemical Company, St. Louis, MO), calf intestine alkaline phos(cid:173)
`phatase (Boehringer Mannheim, Indianapolis, IN), orthophosphate (32P04;
`Dupont-New England Nuclear, Wilmington, DE), Empigen (Emp) BB
`detergent (Calbiochem-Novabiochem Corp., La Jolla, CA), collagenase
`type I (Worthington Biomedical Corp., Freehold, NJ), TransformerTM
`mutagenesis kit (CLONTECH Laboratories, Palo Alto, CA), and pow(cid:173)
`dered Lab Diet (PMI Feeds Inc., St. Louis, MO).
`
`Transgene Construct and Generation of
`Transgenic Lines
`The human K18 eDNA ser52 codon (AGC) was mutated to a GCC (ala)
`codon in a pBluescript SK + plasmid using the Transformer kit as recom(cid:173)
`mended by the manufacturer. Confirmation of the mutation was done by
`sequencing both strands of the mutated region. The mutant eDNA was
`then digested with AlwN I to generate a 250-bp fragment that was then
`substituted for the corresponding wild-type segment of exon 1 in a 10-kb
`genomic K18 clone (provided by Dr. Robert Oshima, The Burnham Insti(cid:173)
`tute, La Jolla, CA), exactly as we had done in generating the arg89-7cys
`K18 genomic mutant (25). The 10-kb K18 ser52-7ala genomic DNA was
`injected into pronuclei of fertilized FVB/N mouse eggs. Progeny mice
`carrying the human K18 gene were then chosen, after PCR screening,
`followed by breeding to select for germline transmission using stan~
`dard methods. Two mouse lines (S52A1 and S52A2) that express K18
`ser52-7ala were expanded and used for subsequent studies. The control
`mice that were used (TG2 mice) overexpressed the wild-type 10-kb ge(cid:173)
`nomic K18 (1, 25, 27). PCR screening of mouse tail genomic DNA for the
`presence of human K18 involved amplification of a 270-bp fragment that
`corresponds to the COOH-terminal region of K18. The primers used
`were:(+) 5'-CAGAAGGCCAGCTTGGAGAAC-3' and(-) 5'-ATC(cid:173)
`TCCTGATCCCAGCACGTG-3'. All mice were housed in the same
`room with standard infection control precautions.
`
`Transgenic Mice, Liver and Serum Testing, and
`Partial Hepatectomy
`For all transgenic mouse hepatectomy and liver toxicity experiments, age
`(all >6 wk old, no more than 2 wk difference in age among the various
`groups) and sex-matched mice were weighed just before use. Liver and
`blood were collected after killing the mice using C02 inhalation, then
`bleeding via intracardiac puncture (0.5-1.0 ml). The liver was them imme(cid:173)
`diately harvested. Serum was analyzed from TG2 and S52A1 and S52A2
`control diet-fed mice (8 mice/line), GF-fed TG2 and S52A1 mice (10 mice/
`line), MLR-treated mice (10 mice/line) for creatinine, glucose, total pro(cid:173)
`tein, alkaline phosphatase, albumin, triglycerides, cholesterol, total biliru(cid:173)
`bin, and alanine and aspartate aminotransferases. Partial hepatectomy
`was done by removing the lateral, left, and right median lobes, as de(cid:173)
`scribed (55, 56) from TG2, S52A1 and S52A2 mice (16 mice/per transgenic
`line). Mice were then killed 24 h (4 mice/line), 48 h (6 mice/line), and 72 h
`(6 mice/line) afterwards and the liver:s were processed as below. For the
`hepatectomy experiments, sham-hepatectomized mice (i.e., mice that had
`anesthesia, an abdominal wall and peritoneal incision, exposure of the
`liver then closure of the incisions) from each transgenic line served as con(cid:173)
`trols. Resected livers (from control diet-fed, sham-hepatectomized, post(cid:173)
`hepatectomy, GF-fed, or MLR-administered) were cut into several pieces
`depending on the experiment and used for one or more of the following:
`(a) fixation with 10% formalin followed by paraffin embedding, section(cid:173)
`ing, then hematoxylin and eosin staining (done by Histo-tec Laboratory,
`Hayward, CA), (b) snap freezing in O.C.T. compound, sectioning then fix(cid:173)
`ing briefly in cold acetone for subsequent immunofluorescence staining,
`(c) snap freezing in liquid nitrogen for subsequent biochemical analysis,
`
`The Journal of Cell Biology, Volume 143, 1998
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`2024
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`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2094 - 6/14
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`

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`and (d) cutting into small fragments then metabolic labeling with 32P04 in
`phosphate-free medium. Liver perfusion was performed as described (12)
`using 0.025% collagenase type I, and hepatocyte viability was determined
`using trypan blue exclusion.
`
`by immunoblotting of serial dilutions of total liver tissue homogenates
`using anti-pankeratin or anti-phosphokeratin antibodies. Northern blot
`analysis was done using total liver RNA.
`
`Griseofulvin and Microcystin-LR Experiments
`A pilot experiment was initially done using TG2, S52A1, and S52A2 mice
`that were fed powered diet ±1.25% GF (6 mice/group). Since both S52A1
`and S52A2 mice gave similar results in terms of having a worse histologic
`score upon GF feeding as compared with TG2 mice, we only used the
`S52A 1 for subsequent experiments. Mice (10/line) were then fed control
`or GF-supplemented diet for 17 d followed by isolation, weighing then
`processing of the livers as described above. Alternatively, mice were fed
`control or OF-containing diet for 2, 5, or 8 d (2 mice/line) followed by the
`processing of the livers. For the MLR experiments, pilot experiments
`were carried out to determine the optimal dose that does not cause rapid
`lethality. A dose of 30 J..Lg/Kg was chosen, and as found for the GF experi(cid:173)
`ments, the S52A 1 and S52A2 had a worse histology than the TG2 mice that
`led us to use the S52A 1 line for the subsequent quantitative experiments.
`Mice (10/line) from each group were given 30 J..Lg/~g of MLR intraperito(cid:173)
`neally, followed by processing of the livers after 195 min. MLR was pre(cid:173)
`pared in dimethylsulfoxide, as a 1 mg/ml stock solution, then diluted in
`PBS (10 mM sodium phosphate/0.15 M NaCl, pH 7.4).
`
`Histology Grading and Statistical Analysis
`All histology slides were examined by a pathologist (S.A. Michie) without
`knowledge of the treatment, diet, or transgenic line. Grading of the OF(cid:173)
`related slides was done by counting the number of necrotic cells in a 20X
`field (10 fields per slide) and is presented as a necrosis score. Grading of
`the MLR-related slides was done by estimating the extent of vacuolization
`(vacuolization score) and area of hemorrhage/necrosis (hemorrhage score).
`The vacuole score was estimated by assigning a 0 to 3+ score depending
`on the percentage of vacuole volume as compared with overall hepatocyte
`cytoplasmic volume such that: 0 = <10%, 1 + = 10-29%, 2+ = 30-49%,
`and 3+ = :::::50%. The hemorrhage score was estimated by assigning a 0 to
`3+ score depending on extent of the hemorrhage and necrosis: 0 =none(cid:173)
`crosis, 1 + =small foci (mostly midzonal), 2+ =confluent areas, and 3+ =
`diffuse hemorrhage and necrosis with rare intact islands of hepatocytes.
`All scores and laboratory parameters are expressed as means ±SO. The
`Student's t test and nonparametric Wilcoxon method were used to calcu(cid:173)
`late the statistical significance between the means. Statistical analysis was
`performed using JMP version 3.1 (SAS Institute Inc., Cary, NC).
`
`Keratin Isolation, Immunoprecipitation, and
`Other Methods
`Liver pieces (rv3 X 4 X 4 mm) were used to isolate keratins exactly as de(cid:173)
`scribed previously, either by immunoprecipitation (26) or high salt extrac(cid:173)
`tion (2, 25) except that NaF and sodium pyrophosphate were not added to
`the solubilization buffer. For immunoprecipitation, liver pieces were solu(cid:173)
`bilized in 1% Emp or 1% NP-40 in PBS containing a protease/phos(cid:173)
`phatase inhibitor cocktail (25). Dephosphorylation of keratin immunopre(cid:173)
`cipitates was done with alkaline phosphatase (20 units/J..Ll stock enzyme
`mix) using 20 units in 20 J..Ll of buffer (supplied by the manufacturer with
`the enzyme) for 1 h (30°C) followed by washing 2X with 1% NP-40 in
`PBS. SDS-PAGE was done using 10% acrylamide gels (31). Radiolabel(cid:173)
`ing of NIH-3T3 or HT29 cells, or liver tissue fragments (250 J..LCi/ml) was
`done in 5 ml or 2 ml, respectively, of phosphate free RPMI-1640 media
`containing 10% dialyzed FCS and 1-2% normal medium. Immunoblotting
`(53) was done by transferring immunoprecipitates or total celllysates (sol(cid:173)
`ubilized in 2% SDS/5% glycerol-containing Laemmli sample buffer) to
`polyvinylidene difluoride (PVDF) membranes followed by blotting with
`1:1,000 dilution of anti-keratin antibodies then visualization of the reac(cid:173)
`tive bands using enhanced chemiluminescence. Tryptic phosphopeptide
`mapping of K18 that was purified by immunoprecipitation from 32P04-
`prelabeled detergent solubilized HT29 or transfected NIH-3T3 cells or
`mouse freshly isolated liver fragments was done exactly as described (7).
`IEF of the keratin precipitates (39) was done using a Mini-PROTEAN II
`apparatus (BioRad Laboratories, Cambridge, MA) and an ampholine pH
`mix of 3-10 and 5-7 as recommended by the manufacturer. Immunofluo(cid:173)
`rescence staining was done as described (26).
`Comparative quantitation of keratin protein levels and phosphoryla(cid:173)
`tion was done using densitometric scanning of Coomassie-stained gels, or
`
`Results
`
`Characterization of Transgenic Mice That Overexpress
`Human K18 ser52~la
`To address the physiologic function of keratin phosphory(cid:173)
`lation, we targeted a specific keratin phosphorylation site
`in the context of an intact transgenic mouse. Our hypothe(cid:173)
`sis was that transgene expression of a keratin that is mu(cid:173)
`tated at a particular phosphorylation site may clarify the
`potential role of keratin phosphorylation during mitosis
`and/or in providing protection from drug-induced liver in(cid:173)
`jury. This hypothesis is based on the increase in K8/18
`phosphorylation that is observed in association with a
`variety of cell stresses in mice and cultured cells (26, 47).
`To test this hypothesis, we generated a genomic K18
`ser52~ala mutant and injected it into mouse embryos
`then selected two lines (S52A1 and S52A2). The wild-type
`(WT) 10-kb genomic K18 construct that we used to gener(cid:173)
`ate the point mutation was identical to what we used pre(cid:173)
`viously to generate a genomic K18 arg89~cys, which af(cid:173)
`forded tissue specific overexpression of human K18 that
`paralleled that of the endogenous mouse K18 (25). For
`comparison, and to serve as a control for WT human K18
`overexpression, we used the well-characterized TG2 mice
`that overexpress WT human K18 (1, 25). To isolate the
`overexpressed human keratin, we used mAb L2A1 that
`recognizes human K18 and does not cross-react with
`mouse keratins (25). As shown in Fig. 1 A, mAb L2A1 im(cid:173)
`munoprecipitated nearly equal levels of keratins from liv(cid:173)
`ers of TG2 mice and mice from the two S52A transgenic
`lines. As would be expected, immunoblotting of these pre(cid:173)
`cipitates with the previously described Ab 3055, which
`specifically recognizes an epitope that contains human
`phospho-ser52 (pS52; reference 37), showed binding only
`to K18 from TG2 mice but not to K18 from S52A mice
`(Fig. 1 A).
`The relative levels of keratin expression were compared
`in the livers of the transgenic lines and normal nontrans(cid:173)
`genic FVB/N mice by examining the total keratin pool us(cid:173)
`ing high salt extraction (Fig. 1 B) and total liver homage(cid:173)
`nates (not shown). In TG2 mice, A-/80% of the type I
`keratins (i.e., the total human ectopic and endogenous
`mouse K18) is human K18 whereas in the S52A mice
`A-/70% of the total keratin is human K18. Both S52A lines
`express similar human K18 levels that in turn are slightly
`lower than those noted in TG2 mice. The endogenous lev(cid:173)
`els of mouse K18 in TG2 and S52A mice, relative to wild(cid:173)
`type mice, are decreased in proportion to the increased
`levels of ectopic human K18 such that the total type I ker(cid:173)
`atins remains essentially constant (not shown). Overall
`and upon normalization of protein levels, type II keratin
`levels are also similar in TG2, S52A, and wild-type mice.
`Assignment of individual keratin bands was done by im(cid:173)
`munoblotting with anti-human and -mouse keratin-spe(cid:173)
`cific antibodies (e.g., Fig. 1 B and data not shown). The