INTRODUCTION

Intermediate filaments (IF) form a large group of nuclear andtissue specific cytoplasmic proteins (Lazarides, 1980; Steinertand Roop, 1988; Fuchs and Weber, 1994). Among thecytoplasmic IF proteins, keratins (K) are specifically expressedin epithelial cells (Moll et al., 1982) and have physiologic andclinical relevance since mutations in 14 of the 20 known ‘soft’keratins (K1-K20) are associated with a variety of skin, oral,liver and corneal diseases (reviewed by Fuchs and Coulombe,1992; Steinert and Bale, 1993; McLean and Lane, 1995;Omary and Ku, 1997; Fuchs and Cleveland, 1998). Theepithelial tissue-specific diseases reflect specific expression ofunique keratin complements depending on the epithelial cellsubtype. For example, K8/18 are expressed in simple epitheliaas found in the intestine, liver, and exocrine acinar pancreas,with variable levels of K19 and K20 depending on the cell type(Eckert, 1988; Stasiak et al., 1989; Moll et al., 1990, 1993),and K1/10 are found in upper epidermis while K5/14 are foundin basal keratinocytes (Steinert and Roop, 1988; Fuchs andWeber, 1994).

Although little is understood regarding keratin function, atleast two important biologic roles for these proteins are known.

First, keratins provide an essential protective cell integrity rolefrom a variety of environmental insults in epidermal, cornealand hepatic cells (Fuchs and Cleveland, 1998; Irvine et al.,1997; Omary and Ku, 1997). Second, keratins interact withseveral other abundant cellular proteins such as the cytoplasmicheat shock proteins (Liao et al., 1995a), and the 14-3-3 proteinfamily (Liao and Omary, 1996). These interactions are likelyto regulate the availability and function of the keratinassociated proteins (Ku et al., 1998a). Notably, modulation ofkeratin phosphorylation is a common associated featurefor these two keratin functions. For example, keratinphosphorylation increases dramatically in association withseveral stress modalities in cultured cells and intact transgenicmouse models (Ku et al., 1996, 1998b; Omary et al., 1998),and K18 serine-33 phosphorylation regulates keratin/14-3-3binding during mitosis (Ku et al., 1998a).

Keratin phosphorylation is a highly dynamic modification(Fey et al., 1983; Steinert, 1988; Klymkowsky et al., 1991;Liao et al., 1995b; Toivola et al., 1997) that occurs within theso-called ‘head’ (N-terminal) and/or ‘tail’ (C-terminal) non-α-helical end domains, but not within the central coiled-coil α-helical domain (Ku et al., 1996; Omary et al., 1998). The likelysignificance of this specific localization of keratin (and all other

2081Journal of Cell Science 112, 2081-2090 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0473

Glandular epithelia express the keratin intermediatefilament (IF) polypeptides 8, 18 and 19 (K8/18/19). Theseproteins undergo significant serine phosphorylation uponstimulation with growth factors and during mitosis, withsubsequent modulation of their organization and interactionwith associated proteins. Here we demonstrate reversibleand dynamic tyrosine phosphorylation of K8 and K19, butnot K18, upon exposure of intact mouse colon or culturedhuman cells to pervanadate. K8/19 tyrosine phosphorylationwas confirmed by metabolic 32PO4-labeling followed byphosphoamino acid analysis, and by immunoblotting withanti-phosphotyrosine antibodies. Pervanadate treatmentincreases keratin solubility and also indirectly increasesK8/18 serine phosphorylation at several known sites, someof which were previously shown to be associated with EGF

stimulation, extracellular signal-regulated kinase (ERK),or p38 kinase activation. However, K8/19 tyrosinephosphorylation is independent of EGF signaling or ERKactivation while inhibition of p38 kinase activity blockspervanadate-induced K8/19 tyrosine phosphorylation. Ourresults demonstrate tyrosine phosphatase inhibitor-mediated in vivo tyrosine phosphorylation of K8/19, but notK18, and suggest that tyrosine phosphorylation may be ageneral modification of other IF proteins. K8/19 tyrosinephosphorylation involves a pathway that utilizes the p38mitogen-activated protein kinase, but appears independentof EGF signaling or ERK kinase activation.

Key words: Keratin, Tyrosine phosphorylation, Pervanadate, p38kinase

SUMMARY

Pervanadate-mediated tyrosine phosphorylation of keratins 8 and 19 via a p38

mitogen-activated protein kinase-dependent pathway

Li Feng1,2,*, Xiangjun Zhou1,2, Jian Liao3 and M. Bishr Omary1,2,‡

1Dept of Medicine, VA Palo Alto Health Care System, 3801 Miranda Avenue, Mail code 154J, Palo Alto, CA 94304, USA2The Digestive Disease Center, Stanford University, MSLS Room P304, Stanford, CA 94305-5487, USA3Clontech Laboratories, Palo Alto, CA 94303, USA*Author to whom reprint requests should be addressed‡Author for correspondence

Accepted 30 April; published on WWW 10 June 1999

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IF protein) phosphorylation is that the ‘head’ and ‘tail’domains impart most of the structural heterogeneity, and hencetissue specific expression and functions, and suggests thatphosphorylation plays an important role in regulating keratinfunction(s) (Ku et al., 1996; Omary et al., 1998; Inagaki et al.,1996). To date, serine (ser) is known to be the exclusive keratinphosphorylation site as demonstrated for K1, K8, K18, andK19 (Gilmartin et al., 1980; Oshima, 1982; Steinert et al.,1982; Chou and Omary, 1991; Zhou et al., 1999), with limitedthreonine phosphorylation that is detected in epidermalkeratins (Steinert et al., 1982, 1988). In contrast, in vivotyrosine phosphorylation of keratins and other IF proteinsappears to be a far less common modification that has not beenas well characterized as serine phosphorylation. For example,barely detectable phospho-tyrosine levels have been describedin crude cytoskeletal epidermal keratin preparations (Steinertet al., 1982), and low levels of keratin tyrosine phosphorylationwere found in pig epidermis crude keratin isolates uponexposure to epidermal growth factor (EGF) (Aoyagi et al.,1985). Vimentin was also reported to undergo tyrosinephosphorylation in lymphoid cells upon exposure to platelet-derived growth factor, as determined using anti-phospho-tyrosine (anti-pY) antibodies, but direct biochemicalconfirmation of phospho-tyrosine was not examined(Valgeirsdottir et al., 1998). In addition, the carboxy-terminaltyrosine of the IF protein peripherin may undergo tyrosinephosphorylation as determined by mutational analysis and anti-pY antibody reactivity, although evidence for modulation ofthis phosphorylation was not reported (Angelastro et al., 1998).

In this study, we used pervanadate (PV) (Fantus et al., 1989;Huyer et al., 1997), a potent tyrosine phosphatase inhibitor, todemonstrate tyrosine phosphorylation of K8 and K19 but notK18 in several cultured human cell lines and in intact mousecolon. Exposure of cells to pervanadate (PV), but not tookadaic acid (OA) (a potent serine/threonine phosphataseinhibitor; Bialojan and Takai, 1988), resulted in K8/19 tyrosinephosphorylation. PV exposure also increased keratin serinephosphorylation at several already characterized sites includingK8 serine-73 (ser73), which is phosphorylated by p38 kinase(unpublished observations) of the mitogen-activated protein(MAP) kinase superfamily (reviewed by Nishida and Gotoh,1993; Davis, 1994; Blumer and Johnson, 1994; Kyriakis andAvruch, 1996). PV-stimulated K8/19 tyrosine phosphorylationinvolves a p38-associated kinase pathway which suggests thatone or more physiologic modalities that activate p38 are likelyto result in K8/19 tyrosine phosphorylation. K8 and K19tyrosine phosphorylation suggests that IF protein tyrosinephosphorylation may be a general modification in uniquesettings. Detection of this phosphorylation using tyrosinephosphatase inhibition suggests that it is highly dynamic.

MATERIALS AND METHODS

Reagents and antibodiesThe cell lines HT29 (human colon) and TE7 (human esophagusadenocarcinoma) (Nishihira et al., 1993) were obtained from theAmerican Type Culture Collection and from Dr T. Nishihira (TheSecond Departmant of Surgery, Tohoku Universiry, Japan),respectively. The antibodies (Ab) used were: mouse monoclonalantibody (mAb) L2A1 which recognizes K18 (Chou et al., 1992);

mAb 4.62 (Sigma, St Louis, MO) which recognizes K19; and anti-phospho-keratin antibodies 5B3, which recognizes K8 phospho-ser431 (pS431) (Ku and Omary, 1997); LJ4, which recognizes K8pS73 (Liao et al., 1997); and Ab 8250, which recognizes K18 pS33(Ku et al., 1998a). The anti-phospho-tyrosine (pY) antibodies usedwere: anti-pY mAb (Clontech Laboratories, Inc., Palo Alto, CA); anti-pY mAb PY66 (Sigma); and rabbit anti-pY Ab and PY-20 mAb(Zymed, San Francisco, CA). Other reagents used were: sodiumvanadate (Fisher Scientific, Pittsburgh, PA), PD98059 (New EnglandBiolabs Inc., Beverly, MA), SB203580 (kindly provided by Dr JohnLee, SmithKline Beecham Pharmaceuticals, King of Prussia, PA),Toto-3 iodide (Molecular Probes, Eugene, OR), EGF (LifeTechnologies, Gaithersburg, MD), Empigen BB (Calbiochem-Novabiochem Corp., La Jolla, CA), and ortho-phosphate (32PO4)(Dupont-New England Nuclear, Wilmington, DE).

Pervanadate treatmentA stock of sodium pervanadate (PV) was prepared by mixing sodiumvanadate in phosphate-buffered saline (PBS) with 30% H2O2, andused within 1 hour. PV (1 ml of 5 mM vanadate/50 mM H2O2) wasinjected rectally, using a blunt end 18G needle, into 2-month-old pre-sedated transgenic mice which overexpress wild-type human K18(termed TG2 mice; Ku et al., 1995). The rectum was then sealed usingsuperglue for the duration of the experiment. The TG2 mice have anormal phenotype and were used in order to facilitate efficientimmunoprecipitation of K8/18/19 using mAb L2A1 (Ku et al., 1995).Mice were killed by CO2 inhalation followed by resection of the colonand rectum in one piece which was divided into 3 segments forhematoxylin and eosin staining, immunofluorescence staining, and forimmunoprecipitation.

Cells were cultured in the presence of PV (50 µM-5 mM) for 5minutes to 3 hours. Maximal keratin tyrosine phosphorylation wasnoted after 60-90 minutes of PV incubation (not shown) and theconcentration of PV used for all experiments was 1 mM except forthe kinase inhibition experiments which used PV at 200 µM. PVexposure (1 mM) resulted in gradual displacement of the cells fromthe dish such that ~50% of the cells become viable floaters after 3hours of exposure to PV. There was no effect on cell viability for atleast 3 hours of PV exposure as determined by trypan blue exclusion(not shown).

Immunoprecipitation and gel analysisCells were solubilized in 1% Nonidet P-40 (NP40) or Empigen BB(Emp) in PBS (pH 7.4) containing 5 mM EDTA, 0.1 mMphenylmethanesulfonyl fluoride, 10 µM pepstatin A, 10 µM leupeptin,25 µg/ml aprotinin, and 1 mM PV (buffer A) (45 minutes).Alternatively, colonic tissues were homogenized with a Dounce using1% Emp then allowed to solubilize for 45 minutes. Aftercentrifugation, the supernatant was used for keratinimmunoprecipitation with mAbs 4.62 or L2A1. In some cases, cellswere solubilized with 1% NP40 in buffer A directly, followed byEmpigen-solubilization, or were solubilized with 1% Emp directly.The detergent Emp solubilizes a significant component of the‘cytoskeletal’ keratin fraction that is not solubilized by NP40, whilemaintaining antigenicity for antibody recognition (Liao et al., 1996).Immunoprecipitates were analyzed by: (i) SDS-polyacrylamide gelelectrophoresis (PAGE) (Laemmli, 1970), followed by Coomassiestaining or transfer to polyvinylidene difluoride (PVDF) membranesfor immunoblotting, or by (ii) isoelectric focusing (IEF) (Liao et al.,1996), SDS-PAGE, then Coomassie staining or transfer to PVDFmembranes for immunoblotting.

Radiolabeling and phosphoamino acid analysisHT29 cells were labeled for 1 hour with 32PO4 (250 µCi/ml) inphosphate-free RPMI-1640 medium supplemented with 10% dialyzedfetal calf serum and 1% (v/v) normal RPMI-1640 medium, followedby the addition of PBS or PV in the presence of the label (1 hour).

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Labeled cells were then solubilized with 1% Emp followed byimmunoprecipitation of the keratins. Precipitates were analyzed bySDS-PAGE, then radiography and blotting (Towbin et al., 1979). Insimilar labeling experiments, radiolabeled then immunoprecipitatedK8 and K19 were individually cut from Coomassie-stained SDS-PAGE gels and electroeluted from the gel slices. After acetoneprecipitation then HCl hydrolysis, the acid-digested 32PO4-labeledkeratins were subjected to phosphoamino acid analysis as described(Boyle et al., 1991).

Treatment with EGF, SB203580 and PD98059Serum-starved HT29 cells were incubated with EGF (1 µg/ml) forvarious time periods followed by solubilization then keratinimmunoprecipitation. In experiments involving the kinase inhibitorsSB203580 or PD98059, cells were incubated with the indicatedconcentrations of SB203580 or PD98059 (60 minutes, 37°C),followed by the addition of 1 µg/ml EGF (30 minutes, 37°C) or 200µM of PV (1 hour, 37°C). Cells were then solubilized in 1% NP40 inbuffer A followed by immunoprecipitation.

Immunofluorescence and histology stainingColonic tissue was fixed in 10% formalin followed by paraffinembedding, sectioning, then hematoxylin and eosin staining. Forimmunofluorescence staining, cells were allowed to adhere ontocoverslips then further cultured in the absence or presence of PV (60minutes). Cells were fixed with methanol (−20°C, 3 minutes), thenincubated with mAb L2A1, LJ4 or anti-pY (1:40 to 1:100). Cells werewashed and double-stained with the DNA-staining dye TOTO-3iodide (1:20,000) and Texas Red-conjugated goat anti-mouseantibody (1:100). For tissue immunofluorescence staining, mousecolonic tissue was snap frozen in O.C.T. compound, sectioned, fixedin cold acetone (5 minutes) then triple stained using anti-pY Ab, anti-keratin antibodies, and TOTO-3 iodide. Fluorescence images wereanalyzed using a Bio-Rad MRC1024 confocal microscope. Imageswere acquired from a single z-plane (0.45 µm thickness) sequentiallyto eliminate bleedthrough.

RESULTS

Pervanadate exposure of cultured glandularepithelial cells results in K8 and K19 tyrosinephosphorylation and increases keratin solubilityWe tested if keratin tyrosine phosphorylation can be inducedusing tyrosine phosphatase inhibition by sodium pervanadate(PV). As shown in Fig. 1A, incubation of HT29 cells with PVresulted in dramatic tyrosine phosphorylation of many cellularproteins (lanes 7,8) as determined by immunoblotting with ananti-phospho-tyrosine antibody (anti-pY Ab). Precipitation ofK8/18/19 using mAb L2A1 then blotting with anti-pY Abshowed binding to what corresponds to Coomassie stained K8and K19 but not K18 (Fig. 1A; lanes 1,2 and 5,6). Thereactivity of anti-pY Ab with K8 and K19 is specifically relatedto pervanadate action within the exposed cells and is not an invitro artifact since incubation of K8/18/19 precipitates withpervanadate, after isolation from non-pervanadate treated cells,did not result in antibody binding (Fig. 1B, compare lanes 2and 3). Further confirmation that anti-pY Ab binds to K8 andK19 but not K18 was obtained by comparing anti-pY Abblots of mAb L2A1 precipitates (L2A1 preferentiallyimmunopurifies K8/18 with a small amount of K19, Fig. 1C,lanes 1-5) with anti-pY blots of mAb 4.62 precipitates (4.62isolates preferentially K8/19 with a small amount of K18, Fig.1C, lanes 1′-5′) that are isolated from NP40-solubilized and

post-NP40 Emp-solubilized cell extracts. The reactivity ofanti-pY Ab with keratins is specific to tyrosine phosphataseinhibition and is not affected by okadaic acid (OA)-mediatedserine phosphatase inhibition. As shown in Fig. 1C (lanes 1-3,1′-3′), OA results in dramatic keratin serinehyperphosphorylation in association with a migration shift ofthe keratin bands on one-dimensional SDS-PAGE gels, but noreactivity with the anti-pY Ab. Three other commerciallyavailable independent anti-pY antibodies (see Materials andMethods) also gave similar reactivity with K8/19 precipitatesafter PV treatment (not shown). The anti-pY Ab recognizesboth K8 and HK8 (Fig. 1A, lanes 2,6) without any apparentselectivity, and anti-pY Ab binding to K8/19 is reversible inthat it is abolished 24 hours after washing off the PV (notshown).

Confirmation that anti-pY Ab binding to K8/19 after PVtreatment represented specific tyrosine phosphorylation wasobtained in three ways. First, we blotted keratin precipitateswith anti-pY Ab after separation of the precipitates by 2-dimensional gels. As noted in Fig. 2c, the anti-pY Ab did notbind any K18 isoforms but did react strongly with the acidicisoforms of K8 and K19. This indicates that keratin reactivitywith anti-pY Ab represents binding to charged (i.e. most likelyphosphorylated) isoforms. Second, we directly tested K8/19tyrosine phosphorylation by 32PO4 metabolic labeling of thekeratins in cells that were cultured in the presence or absenceof PV. Incubation of HT29 cells with PV increased K8/18/1932PO4 incorporation (Fig. 3A), and caused an acidic shift of thekeratin isoforms (Fig. 2a,b). In addition, phosphoamino acidanalysis of K8 and K19 showed tyrosine phosphorylationupon tyrosine phosphatase inhibition (Fig. 3B), which isundetectable if phosphoamino acid analysis is done on K8 andK18 (Chou and Omary, 1991, 1993) or K19 (Zhou et al., 1999)that are isolated from cells cultured in the absence of PV. Therelative stoichiometry of K19 tyrosine phosphorylation issignificantly higher than that of K8, based on phosphoaminoacid analysis (Fig. 3B) and the relative blotting intensity ofanti-pY Ab binding to K8 versus K19 (Fig. 1C and Fig. 3A).Third, treatment of K8/19 immunoprecipitates, that areobtained from PV-treated HT29 cells, with alkalinephosphatase results in near abolishment of anti-pY Abreactivity (not shown).

We also examined the effect of cell exposure to PV onkeratin solubility, given that such exposure resulted insignificant hyperphosphorylation of K8/18/19 (Figs 2, 3) andearlier findings that keratin hyperphosphorylation is associatedwith an increase in the soluble keratin compartment (Ku et al.,1996; Omary et al., 1998). As shown in Fig. 3C, PV treatmentincreased keratin solubility in the nonionic detergentextractable fraction with a concomitant decrease in the keratinfraction in the remaining Emp-extractable fraction. After PVtreatment, the tyrosine phosphorylated K19 and K8 specieswere distributed nearly equally among the cytosolic, NP40 andEmp fractions (not shown).

PV exposure of normal mouse colon in situincreases keratin tyrosine phosphorylationThe PV-induced K8/19 (but not K18) tyrosine phosphorylationin colonic HT29 cells was also noted in other tested humanepithelial cell lines including the esophageal cell line TE7which also expresses K8/18/19 (not shown). We examined if

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the PV-induced keratin tyrosine phosphorylation can alsooccur in normal mouse tissues in situ. Previous studies showedthat mouse intraperitoneal injection of PV resulted in tyrosinephosphorylation of several liver signaling proteins (Ruff et al.,1997). For this experiment we injected PV rectally as an enemainto mice, then examined total cell protein and K8/18/19tyrosine phosphorylation. We chose rectal administration sincenormal colonic epithelial cells express K8/18/19 whilehepatocytes express only K8/18 (Osborn et al., 1986). Asshown in Fig. 4A, in situ exposure of mouse colon to PVresulted in significant tyrosine phosphorylation of severalproteins. K19 and K8 tyrosine phosphorylation also increasedsignificantly (Fig. 4A) as noted in the cultured cells (e.g. Fig.

1). Exposure of mouse colon to PV resulted in severalprominent histopathological findings including edema,sloughing of the tip mucosal cells into the lumen, and vascularcongestion (Fig. 4B).

Comparison of the immunofluorescence staining of mousecolon before and after exposure to PV showed a dramaticincrease in pY Ab staining, as expected, which was coupledwith neo-phosphorylation of K8 ser73 (Fig. 5). The increase inK8 ser73 phosphorylation occured over the entire epithelium(Fig. 5b,e) while the increase in pY staining occured initiallyin the villus tips (Fig. 5, compare a and d) then migrated inwardtowards the crypt with increasing PV incubation time (notshown). As described previously, K8 ser73 phosphorylation

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Fig. 1. Anti-phosphotyrosineantibodies bind to K8/19 aftertreatment of HT29 cells with PV.(A) HT29 cells were cultured in thepresence or absence of 1 mM PV(90 minutes) followed bysolubilization with 1% NP40 thenprecipitation using mAb L2A1.Keratin immunoprecipitates (i.p.)and detergent lysates were analyzedby SDS-PAGE then Coomassiestaining, or were transferred into aPVDF membrane then blotted withanti-pY Ab. Note that PV treatmentgenerates a hyperphosphorylatedform of K8 (HK8) due toindependent serine phosphorylationof K8 at ser73 (see also C). Thebands in lane 6 that are labeled ‘a’and ‘b’ correspond to HK8/K8 andK19, respectively, as determined byCoomassie staining of the blottedmembrane (not shown).(B) K8/18/19 precipitates wereobtained from HT29 cells that werecultured in the absence (lane 1) orpresence of 1 mM PV (lane 3) as inA. Alternatively, an identicalprecipitate to that shown in lane 1was incubated (after precipitation)with 1 mM PV for 90 minutesfollowed by washing (lane 2).Precipitates were analyzed by SDS-PAGE then Coomassie staining, andby blotting with anti-pY Ab.(C) HT29 cells were cultured for 90minutes in the presence, or absence,of PV (1 mM) or okadaic acid(1 µg/ml), followed by sequentialsolubilization with 1% NP40 then1% Emp as described in Materialsand Methods. Detergent lysateswere used to preferentiallyimmunoprecipitate K8/18 (usingmAb L2A1) or K8/19 (using mAb4.62). Note that a small amount ofK19 or K18 coprecipitates with K8/18 or with K8/19, respectively, due to the heteropolymeric nature of keratins. Precipitates were blotted withanti-pY Ab or with Abs that recognize K18 phospho-ser33 (pS33) or K8 pS73. The band just below HK8/K8 (seen most prominently in lanes2′ and 3′) corresponds to uncharacterized tyrosine phosphorylated protein(s), indicated by an asterisk in the Coomassie stained gel, thatcoprecipitate(s) with K8/18/19. Note that the anti-pY Ab reacts considerably more strongly with K19 than with K8 (e.g. compare lanes 2 with2′, and lanes 2′ with 5′, which show changes in anti-pY Ab binding to K8 and K19 depending on their Coomassie blue staining intensity).

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occurs under a variety of conditions including cell stress,mitosis and apoptosis (Liao et al., 1997), and was also notedin cultured HT29 cell after exposure to PV (Fig. 1). Theincreased binding of anti-K8 ser73 mAb LJ4, which recognizesthe pS73 K8 site (pS73 K8 is conserved in mouse and humanK8; Liao et al., 1997), upon PV exposure of mouse colon wasconfirmed by blotting of the K8/18/19 immunoprecipitateswith mAb LJ4 and specific recognition of the mouse HK8species (not shown). Of note, immunofluorescence staining ofcultured HT29 cells after PV treatment showed somereorganization of the keratin filaments (not shown). However,specific association of such reorganization with tyrosinephosphorylation cannot be ascertained given the concomitantincrease in keratin serine phosphorylation (e.g. Fig. 1).

Probing of the signaling pathway of PV-inducedK8/19 tyrosine phosphorylationSince an earlier report suggested that EGF may promote thetyrosine phosphorylation of epidermal keratins (Aoyagi et al.,1985), we tested the effect of EGF on K8/19 tyrosine

phosphorylation in cultured HT29 cells and also probedpotential intracellular pathways that may play a role in theobserved effect of PV on keratin tyrosine phosphorylation. As

Fig. 2. Two-dimensional gel analysis of keratins that are isolated from PV-treated and untreated cells. K8/18/19 immunoprecipitates wereobtained, using mAb L2A1, from HT29 cells that were cultured in the presence or absence of PV (1 mM, 90 minutes). Precipitates were thenanalyzed by IEF (first dimension), SDS-PAGE (second dimension), followed by Coomassie staining (a,b). A duplicate gel to that shown inpanel b was blotted with anti-pY Ab (c). Numbers indicate identical isoforms based on mixing experiments (not shown). Note the acidic shift ofthe keratin isoforms after pV treatment, and that the isoforms that are recognized by the anti-pY Ab correspond to the acidic (i.e. likelyphosphorylated) isoforms.

Fig. 3. Keratin 32PO4 labeling, phosphoamino acid analysis, andsolubility after pervanadate exposure of HT29 cells. (A) HT29 cellswere labeled with 32PO4 (2 hours, 250 µCi/ml) in the presence orabsence of 1 mM PV for the last 1 hour of labeling. Cells were thensolubilized with 1% Emp, and the detergent lysate was divided intotwo equal parts. One part was used to preferentially precipitateK8/18 (mAb L2A1) while the second part was used to precipitateK8/19 (mAb 4.62). Immunoprecipitates were analyzed by SDS-PAGE, Coomassie staining, autoradiography, then were blotted withanti-pY Ab. (B) K8 and K19 were individually purified from PV-treated 32PO4-labeled HT29 cells, then subjected to phosphoaminoacid analysis as described in Materials and Methods. The arrowsshow the position of 32PO4-labeled phospho-serine (pS) andphospho-tyrosine (pY). The dotted arrow indicates the position ofphospho-threonine (pT) which is not metabolically labeled in K8 orK19 (or K18, not shown). (C) Equivalent numbers of cells werecultured in the presence or absence of PV (90 minutes, 1 mM)followed by sequential solubilization with 1% NP40, then 1% Emp,then precipitation of K8/18/19 with mAb L2A1 under antibodysaturating conditions. Equivalent fractions of the precipitates wereanalyzed by SDS-PAGE then Coomassie staining. Identity of theband highlighted by an asterisk is unknown.

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shown in Fig. 6A, exposure of HT29 cells to EGF resulted ina time dependent increase in K8 ser431 and ser73phosphorylation, as described previously for K8 ser431 (Kuand Omary, 1997), but did not affect keratin tyrosinephosphorylation (lanes 1-5). The EGF-mediated increase in K8ser431 phosphorylation was inhibited by preincubation ofHT29 cells with the extracellular-regulated kinase kinaseinhibitor PD98059, which in contrast did not affect basalbinding of the anti-pY Ab with K8 or K19 (Fig. 5A, lanes 5-7). Given that keratin tyrosine phosphorylation is associatedwith increased K8 ser73 phosphorylation (which generates the‘HK8’ species, e.g. Fig. 1), and that the mitogen-activatedprotein kinase p38 appears to be an important kinase for K8ser73 phosphorylation (unpublished observations), we asked ifp38 kinase inhibition plays a role in the PV-associated K8/19

tyrosine phosphorylation. As shown in Fig. 6B, PD98059 hadno significant effect on PV-induced keratin phosphorylation,while the p38 kinase inhibitor SB203580 dramatically bluntedPV-induced K8 and K19 tyrosine phosphorylation in a dose-dependent fashion. This suggests that a p38-like kinase-mediated signaling cascade plays a direct or indirect role in theobserved K8 and K19 tyrosine phosphorylation.

DISCUSSION

Characterization of K8/19 tyrosine phosphorylationThis study presents immunologic and direct biochemicalevidence for tyrosine phosphorylation of K8 and K19 but not

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Fig. 4. Tyrosine phosphorylation of keratinsisolated from PV-treated mouse colon.(A) Mice were given 1 ml of 5 mM PV inPBS rectally for 0, 15, 30, or 75 minutesfollowed by resection of the colon thenhomogenization. Detergent lysates wereanalyzed directly by SDS-PAGE or wereused for precipitation of K8/18/19 (mAbL2A1). Precipitates or aliquots of thedetergent lysates were used for blotting withanti-pY Ab. Note that the pY Ab reacts withan unknown endogenous protein that is seenin all lanes 1′-4′ (indicated by a singleasterisk), while tyrosine-phosphorylated K19(which migrates just below the bandhighlighted by a single asterisk) becomesprogressively visible with increasing PVexposure times. The identity of K19 wasconfirmed by blotting of K8/19 precipitatesthat were separated by 2-dimensionalIEF/SDS-PAGE as done in Fig. 2 (notshown). The identity of the band that isindicated by a double asterisk is not known.(B) Pieces of colon were isolated fromuntreated or PV-treated (75 minutes) mice,fixed then stained with hematoxylin andeosin. Arrows indicate the lumen (L), edemabetween the crypts (E), and vascularcongestion (V). Note cell sloughing into thelumen in histogram b.

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of K18, which in turn provide support for the selectivity andspecificity of this modification. It also supports suggestionsfrom early studies that epidermal keratins may be tyrosinephosphorylated upon EGF stimulation although in thesestudies the keratins consisted of crude preparations (Aoyagi etal., 1985). The evidence for K8/19 tyrosine phosphorylationincludes: (i) binding of multiple anti-phosphotyrosineantibodies to these keratins when cells are stimulated with thetyrosine phosphatase inhibitor pervanadate but not with theser/thr phosphatase inhibitor okadaic acid (Fig. 1); (ii) bindingof anti-pY Ab, after 2-dimensional isoelectric focusing/SDS-PAGE separation, exclusively to the acidic isoforms of K8 andK19 but not to the relatively basic unphosphorylated isoforms(Fig. 2); (iii) detection of phospho-tyrosine in K8 and K19after metabolic labeling of PV-treated HT29 cells with 32PO4(Fig. 3); and (iv) inhibition of anti-pY Ab binding to K8/19(that is isolated from PV-treated cells) upon treatment withalkaline phosphatase (not shown). Binding of K8 and K19 toanti-pY Ab was also noted in several human epithelial celllines, and in mouse colon in situ upon exposure to pervanadate(Fig. 4). In the case of mouse colon, keratin tyrosinephosphorylation was observed preferentially in K19 (ascompared with K8), but this is consistent with what is seen inhuman HT29 cells in that the stoichiometry of K19phosphorylation is significantly greater than that of K8 (Fig.1 and Fig. 3B). The tyrosine phosphorylation of K8 and K19

but not K18 also provides evidence for the selectivity andspecificity of this modification.

Few suggestions of tyrosine phosphorylation of other IFproteins have also been described including in vitrophosphorylation of desmin by the Src kinase (Collet et al., 1980),and phosphorylation of vimentin in porcine aortic endothelialcells upon stimulation with platelet-derived growth factor(Valgeirsdottir et al., 1998). In the latter case, vimentin tyrosinephosphorylation was determined by anti-pY Ab binding but wasnot confirmed by phosphoamino acid analysis or blotting ofisoforms that are separated by isoelectric focusing. Confirmationof tyrosine phosphorylation by more than one modality may beimportant since we find in some cases nonspecific binding of theanti-pY antibodies to the basic keratin isoforms upon separationby IEF (Ku et al., 1996). Rat peripherin tyrosine phosphorylationwas also described (Angelastro et al., 1998), but thisphosphorylation shows several unusual features that may beunique to peripherin. For example, anti-pY blotting of peripherinshows reactivity with all the peripherin isoforms which impliessignificant basal constitutive tyrosine phosphorylation, anobservation that has not been described for any IF protein. Theidentification of the most terminal peripherin residue (Y474) asaccounting for all peripherin tyrosine phosphorylation raises thequestion of whether the free carboxyl group of this tyrosine maymimic a phosphate and thereby possibly crossreact with the anti-pY antibody.

Fig. 5. Immunofluorescence staining of PV-treated or untreated mouse colon. Pieces of colon that were isolated from 30 minutes PV-treated(d-f) or untreated (a-c) mice were triple stained with anti-pY Ab (a,d), anti-K8 pS73 mAb (b,e), and TOTO-3 iodide which stains nuclei (c,f).Fluorescent images are shown in black and white but were collected using a confocal microscope and channels that corresponded to red (pY),green (K8 pS73) or blue (nuclear) staining. Note that the K8 pS73 staining, which is absent except for few basal and tip cells in the −PVspecimen, covers the entire gland after exposure to PV. In contrast, pY is initially ‘relatively’ undetectable in the absence of PV then appearsprimarily at the tip and upper region of the crypts after exposure to PV. L, lumen.

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The potential significance and kinase pathway ofK8/19 tyrosine phosphorylationPervanadate treatment of HT29 cells resulted not only inkeratin tyrosine phosphorylation, but also indirectly affectedkeratin serine phosphorylation. One effect of HT29 cellexposure to PV was increased keratin solubility (Fig. 3C).Since increased keratin solubility may be caused solely byincreased keratin serine phosphorylation (Ku et al., 1996;Omary et al., 1998) independent of any tyrosinephosphorylation, it is unclear at this stage if tyrosine

phosphorylation per se plays any role in modulating keratinsolubility and partitioning into membrane and/or cytosoliccompartments. The PV-mediated changes in keratin serinephosphorylation are unique in that they generate distinctcytosolic, membrane-associated (i.e. NP40 soluble) andcytoskeletal (NP40 resistant but Emp soluble) keratin serinephosphorylation states (not shown) that are different from whatis seen after a variety of other modulations of keratinphosphorylation including those caused by ser/thr phosphataseinhibition or anti-mirotubule agents (Ku et al., 1996; Omary etal., 1998). Therefore, the PV-associated increase in keratinsolubility appears to be unique and distinct from otherconditions that are associated with increased keratin solubility.

The kinetics of keratin tyrosine phosphorylation uponexposure of HT29 cells to PV show that keratin tyrosinephosphorylation becomes detectable nearly 30 minutes afterexposure, while the increase in K8 ser73 (and of several otherunknown cellular proteins) phosphorylation occurs sooner(≤15 minutes, not shown). The antecedent response of K8ser73 phosphorylation, as compared with keratin tyrosinephosphorylation, was also noted in mouse colonic tissues (Fig.4 and not shown). However, K8 ser73 phosphorylation per seis not sufficient to signal tyrosine phosphorylation since severalother conditions that are associated with significant K8 ser73phosphorylation such as apoptosis, a variety of other cellstresses, culturing in the presence of OA (Fig. 1C), and mitosis(Liao et al., 1997) do not result in any detectable keratintyrosine phosphorylation (not shown). In the HT29 cell systemwe used, the PV-induced generation of floater cells (seeMaterials and Methods) occurs quickly and as such is similarto the rapid effect of cell rounding and detachment that isinduced by okadaic acid independent of apoptosis (not shown).Our overall findings suggest that keratin tyrosinephosphorylation is not only somewhat difficult to detect, butalso is likely to involve unique physiologic stimuli and not tobe a prevalent modification.

Modulation of keratin serine phosphorylation in associationwith PV treatment suggested that pathways that involve suchserine phosphorylation may play a role in the observed keratintyrosine phosphorylation. Two such serine phosphorylationsites are K8 ser431, which is phosphorylated by the ERK1/2MAP kinases (Ku and Omary, 1997), and K8 ser73 which isphosphorylated by mitogen-activated p38 kinase (unpublishedobservations). Inhibition of PV-induced keratin tyrosinephosphorylation by SB203580 but not by PD98059 (Fig. 5B)indicates that modulation of the p38 MAP kinase pathway, butnot the ERK1/2 kinase pathway, is associated with K8/19tyrosine phosphorylation. To that end, activation of p38 kinaseby PV was reported previously in baboon smooth muscle cells(Daum et al., 1998). In addition, activation of MAP kinasessuch as ERK1/2, p38, and JNK by peroxyvanadiumcompounds, independent of MAP kinase kinase activation, hasbeen shown in rat hepatocyte cultures (Band and Posner, 1997).The lack of a PD98059 effect on PV-stimulated keratin tyrosinephosphorylation is also supported by the absence of an EGFeffect on keratin tyrosine phosphorylation in the HT29 cellsystem, while PD98059 did inhibit EGF stimulated K8 serinephosphorylation (Fig. 5A). Potential physiologic contexts toconsider, that are associated with p38 kinase activation andmay involve keratin tyrosine phosphorylation, includenumerous signals such as growth factors, inflammatory

L. Feng and others

Fig. 6. Effect of EGF and PV on keratin phosphorylation in thepresence or absence of extracellular-regulated kinase kinase and p38kinase inhibitors. (A) HT29 cells were cultured in serum-freemedium for 24 hours followed by the addition of EGF (1 µg/ml) for15, 30, 60, or 90 minutes (lanes 1-5). Duplicates of the 60 and 90minutes EGF-treated cells were also prepared with the addition ofthe extracellular-regulated kinase kinase inhibitor, PD98059 (100µM), 1 hour before the addition of EGF (lanes 6,7). Cells were thenprocessed for K8/18/19 precipitation followed by SDS-PAGE thenCoomassie staining or immunoblotting. (B) Cells were cultured inthe presence or absence of PV (200 µM, 1 hour). PV-treated cellswere preincubated for 1 hour with DMSO carrier (0.1%, v/v),PD98059 (50-250 µM) or SB203580 (10-150 µM), followed byimmunoprecipitation of K8/18/19. Precipitates were analyzed bySDS-PAGE then Coomassie staining or were blotted with anti-pYAb.

2089Keratin tyrosine phosphorylation

cytokines, apoptosis, a variety of stresses (e.g. heat, UV,chemical/drug, oxidative and osmotic stresses) and serumstarvation. This apparent nonselectivity in p38 kinaseactivation may be segregated by an emerging group of scaffoldand adapter proteins that regulate the activity of MAP kinases(reviewed by Whitmarsh and Davis, 1998; Schaeffer andWeber, 1999).

PV can induce cells to undergo a variety of biologic changesdepending on the cell system tested and the concentration ofthe vanadium compound utilized. These changes includeinduction of differentiation or proliferation, insulin-like effects,and apoptosis (reviewed by Morinville et al., 1998). The likelymechanism of these biologic effects is the inhibition ofphosphatases that appear to play a major role in regulating theactivity of several MAP kinase family members, including p38kinase (reviewed by Keyse, 1998). However, the effect of PVmay also be indirect since vanadate compounds can cleaveproteins (e.g. tubulin; Correia et al., 1994) and DNA (Hiort etal., 1995) in a photoactivation-dependent fashion.

Although K8 and K19 amino acid sequences do not havetypical consensus sequences for receptor or cytosolic tyrosinekinases, identification of the keratin tyrosine phosphorylationsites may shed light on the kinases that may be involved. Tothat end, Yes kinase associates with vimentin IF proteins(Yoshinaka et al., 1995; Ciesielski-Treska et al., 1995), and Srckinase family members, which include the Yes kinase, may playa role in phosphorylating vimentin on tyrosine (Valgeirsdottiret al., 1998). In the case of K8/18/19 immunoprecipitates thatare obtained after exposure of HT29 cells to PV, we did notdetect an association between K8/18/19 precipitates and Yes orSrc kinases (not shown). Further characterization of keratin andother potential IF protein tyrosine phosphorylation,examination of physiologic contexts that involve p38 MAPkinase activation and keratin tyrosine phosphorylation, and theemerging data regarding IF protein function should provideinformation regarding its precise biologic role.

We are grateful to Evelyn Resurreccion for assistance with tissuesectioning and fluorescene staining; Diana Toivola for assistance withconfocal microscopy; Kris Morrow for preparing the figures; SteveAvolicino (Histo-Tec Laboratory, Hayward, CA) for the histologystaining; Sara Michie for assistance with colon histology; John Lee(SmithKline Beecham Pharmaceuticals) for supplying SB203580, andRomola L. Breckenridge for assistance with word processing. Thiswork was supported by a Veterans Affairs Career DevelopmentAward, NIH Grant DK52951, and Digestive Disease Center GrantDK38707.

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