(Received for publication, February 12, 1997, and in revised form, April 2, 1997)
From the Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304 and the Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305-5487
Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. We previously showed that several types of cell stress such as heat and virus infection result in a distinct hyperphosphorylated form of K8 (termed HK8). To better characterize K8/18 phosphorylation, we generated monoclonal antibodies by immunizing mice with hyperphosphorylated keratins that were purified from colonic cultured human HT29 cells pretreated with okadaic acid. One antibody specifically recognized HK8, and the epitope was identified as 71LLpSPL which corresponds to K8 phosphorylation at Ser-73. Generation of HK8 occurs in mitotic HT29 cells, basal crypt mitotic cells in normal mouse intestine, and in regenerating mouse hepatocytes after partial hepatectomy. Prominent levels of HK8 were also generated in HT29 cells that were induced to undergo apoptosis using anisomycin or etoposide. In addition, mouse hepatotoxicity that is induced by chronic feeding with griseofulvin resulted in HK8 formation in the liver. Our results demonstrate that a "reverse immunological" approach, coupled with enhancing in vivo phosphorylation using phosphatase inhibitors, can result in the identification of physiologic phosphorylation states. As such, K8 Ser-73 phosphorylation generates a distinct HK8 species under a variety of in vivo conditions including mitosis, apoptosis, and cell stress. The low steady state levels of HK8 during mitosis, in contrast to stress and apoptosis, suggest that accumulation of HK8 may represent a physiologic stress marker for simple epithelia.
Epithelial cells express the keratin subfamily of intermediate filament (IF)1 proteins in a cell-type preferential manner (1). More than 20 unique keratin gene products (K1-K20) are characterized and classified into type I (K9-K20) and type II (K1-K8) IFs. This classification has biologic relevance since epithelial cells express in a tissue preferential manner at least one type I and one type II keratins that form noncovalent obligate heteropolymers in a soluble tetrameric form or a more complex filamentous cytoskeletal form (2, 3). For example, glandular simple type epithelia express K8 and K18 (K8/18) with variable levels of K19 and K20, and stratified epithelia express K5/14 basally and K1/10 suprabasally. Although the function of IF proteins, including keratins, remains poorly understood, their importance in human disease is accumulating. For example, mutations in epidermal type keratins (e.g. K5/14, K1/10) result in several blistering and scaling skin diseases (4-6). More recently, a mutation in K18 that is associated with an in vitro assembly defect was identified in a patient with chronic liver disease of "unknown etiology" (7), thereby suggesting a predisposition or cause of the patient's cryptogenic cirrhosis (8).
Although IF protein functions remain poorly understood, characterization of the dynamic and regulatory modification of phosphorylation is beginning to provide important functional clues (9, 10). A functional and regulatory role for IF protein phosphorylation is supported by the location of this modification within the N-terminal "head" and C-terminal "tail" domains of IF proteins which are the domains that impart most of the structural heterogeneity and hence tissue-specific expression of these proteins. In the case of K8/18, phosphorylation regulates filament reorganization in vivo, enhances keratin solubility, plays a role in dictating the localization of K8/18 with specific cellular compartments, regulates the association with the 14-3-3 protein family, and is associated with a variety of physiologic stresses (10). With regard to cell stress, several stress modalities have been associated with hyperphosphorylation of K8/18 including heat stress and virus infection (11), and mitotic arrest (which can be considered a form of stress) of several cultured cell lines (12). Also, drug-induced hepatotoxic stress, induced in mice by feeding with a griseofulvin-supplemented diet, resulted in dramatic K8/18 hyperphosphorylation (13). Of note, stress-induced K8 hyperphosphorylation is associated with generation of a distinct K8 species, termed HK8 (11, 12), that migrates slightly slower than K8 after one-dimensional gel analysis. To date, two in vivo K8 phosphorylation sites have been described (14). One site, Ser-431, is located in the tail domain of K8 and is phosphorylated after stimulation of cells by epidermal growth factor and is likely to be phosphorylated by mitogen-activated protein kinase (MAPK). The second site, Ser-23, is located in the head domain of K8 and is a conserved site in all type II keratins. This latter site is also phosphorylated in K6 and hence is likely to be a conserved phosphorylation site that serves a common function in all type II keratins. Other in vitro phosphorylation sites of K8 that were phosphorylated by purified cAMP-dependent protein kinase have also been described (15).
In this report we used a "reverse immunological" approach, which entailed utilizing several existing strategies, to identify physiologic K8/18 phosphorylation sites. This approach involves generating monoclonal antibodies that selectively recognize phosphorylated K8 or K18, by immunizing mice with keratins that were purified from cultured human cells that were treated with okadaic acid to generate hyperphosphorylated K8/18. One such characterized antibody (LJ4) selectively recognized HK8, and the epitope of the antibody was identified as Ser(P)-73 of K8. The antibody was then used to demonstrate that formation of HK8 occurs in association with several physiologic events including cell stress, apoptosis, and mitosis. The biologic implications of these findings are discussed.
HT29 (human colon) and baby
hamster kidney cultured cells and the cDNA for human K8 were
obtained from the American Type Culture Collection (Rockville, MD).
Transgenic mice that express wild-type human keratin 18 (termed TG2)
have been described previously (16, 17). Antibodies used were mouse
monoclonal antibody (mAb) L2A1 (18), rabbit anti-human K8/18 antibody
8592 (17), and Troma I rat anti-mouse K8 mAb (Developmental Studies
Hybridoma Bank, University of Iowa). The LJ4 and LJ5 antibodies are
described below. Other reagents/supplies were okadaic acid (LC
Services, Woburn, MA), polyvinylidene difluoride (PVDF) membrane
(Millipore, Bedford, MA), Freund's adjuvant, griseofulvin,
aphidicolin, and protein kinase A (Sigma), immobilized protein A and
BCA protein determination kit (Pierce), enhanced chemiluminescence
reagent (Amersham Corp.), protein kinase C (PanVera Corp.,
Madison, WI), calcium/calmodulin-dependent kinase II, p34cdc2
and MAPK (New England Biolabs, Cambridge, MA), anisomycin (Calbiochem), and etoposide (Bristol-Myers, Princeton, NJ).
For okadaic acid (OA) treatment, HT29 cells were incubated with culture medium containing 1 µg/ml OA for 2 h at 37 °C. Heat stress was done by culturing cells (~50% confluent and in log phase) at 42 °C for 24 h. To induce apoptosis, cells were treated with 10 µM anisomycin (16 h) or with 200 µM etoposide for 4 h followed by switching to normal culture medium without drug for 48 h. Cell cycle G2/M stage enriched HT29 cells were generated by arresting cells at G1/S using aphidicolin and then washing off the drug and culturing for 10 h (19). This synchronization typically resulted in 70-90% G2/M enriched cells (not shown). Cell cycle analysis was done as described (19). Griseofulvin (GF) feeding (1.25% w/w) or control diet feeding was done for 17 days exactly as described using Balb/c or TG2 transgenic mice (13). Partial hepatectomy was carried out as described (13) followed by harvesting of the livers for immunoprecipitation and immunostaining. Sham hepatectomies were done in a manner identical to the experimental hepatectomy (i.e. anesthesia, abdominal wall and peritoneal incision, exposure of the liver, and closure of the incisions) except that liver tissue was not resected.
Generation of Phosphoepitope-specific Monoclonal AntibodiesHyperphosphorylated keratins (K8/18/19) were purified by immunoprecipitation, using mAb L2A1-Sepharose-protein A beads, from cultured HT29 cells that were treated with okadaic acid (1 µg/ml, 2 h). The keratins were eluted from the covalently coupled antibody beads using 9 M urea followed by dialysis in PBS. Balb/c mice were immunized intraperitoneally using 20 µg of purified keratins mixed with complete Freund's adjuvant. The immunizations were repeated four times using incomplete Freund's adjuvant at 4-6-week intervals. Three days before fusion (20), mice received a booster (20 µg) via tail vein injection. Hybridoma culture supernatants were screened by Western blotting using detergent lysates of HT29 cells that were cultured in the presence or absence of OA. One hybridoma clone (LJ4) reacted preferentially with OA-treated HT29 cell lysates as compared with non-OA-treated HT29 cells. Cells from hybridoma clones of interest were cloned by limiting dilution. mAb LJ5, which reacted equally with phosphorylated and non-phosphorylated K8, K18, and K19 (not shown), was also selected and cloned. The isotypes of mAbs LJ4 and LJ5 were determined as IgG1.
Immunoprecipitation, Gel, and Blot AnalysisWashed cells were solubilized using 1% Nonidet P-40 or 1% Empigen BB (21) in PBS (pH 7.4) containing 10 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, 10 µM pepstatin A, 10 µM leupeptin, 25 µg/ml aprotinin, and 0.5 µg/ml OA (45 min, 4 °C). After spinning (16,000 × g, 30 min, 4 °C), the supernatants were used for immunoprecipitation (6 h, 4 °C) with antibodies that are covalently conjugated to protein A-Sepharose. To test the blocking effect of phosphorylated and nonphosphorylated synthetic peptides, antibody beads were first incubated with 100 µg of individual peptides in 200 µl of PBS for 1 h followed by the addition of cell lysates (500 µl) and then immunoprecipitation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (22) was performed using 10% polyacrylamide gels under nonreducing conditions. Two-dimensional gel electrophoresis was carried out as described (23). For Western blotting, tissue or cell lysates, immunoprecipitates, or purified keratin proteins were resolved on SDS-polyacrylamide gels and then electrophoretically transferred to PVDF membranes. Western blotting was done as described (19). Dot blots were performed using a nitrocellulose membrane. Peptides (0.5 µg in 1 µl) were spotted on the nitrocellulose membrane followed by immunoblotting as done for the Western blotting except that the blocking buffer consisted of 3% bovine serum albumin in PBS (instead of 5% non-fat milk powder in PBS).
Peptide Synthesis and Enzyme-linked Immunosorbent Assay (ELISA)Peptide synthesis was carried out as described (19) using an automated 9050 Milligen Peptide Synthesizer. The ELISA was done by coating microtiter plates overnight (22 °C) with serial 2-fold dilutions of peptides (0.125 to 4 µg in 100 µl/well) in 100 mM sodium carbonate (pH 9.6). The plates were then rinsed twice with washing buffer (10 mM sodium phosphate (pH 7.2), 0.05% Tween 20). Blocking was performed for 2 h (22 °C) using 200 µl/well of the coating buffer containing 1% bovine serum albumin. After washing, 100 µl/well of mAb (cell culture supernatant diluted 1:5 in washing buffer) were added in the presence or absence of peptides and incubated for 1 h, following by washing four times. Horseradish peroxidase-conjugated rabbit anti-mouse antibody (100 µl/well, 1:1000 dilution in washing buffer) was added (1 h). Wells were then washed four times followed by the addition of 100 µl/well of the peroxidase substrate (30 min) and measuring the absorbance at 405 nm.
In Vitro Phosphorylation and Immunofluorescence StainingK8/18 immunoprecipitates, which contain minimally
phosphorylated K8/18, were obtained from HT29 cells (24). This was done by solubilization with 1% Nonidet P-40, spinning, and then
solubilization of the pellet with 1% Empigen followed by
immunoprecipitation. After heating (95 °C, 1 min) to destroy any
associated kinase activity, precipitates were phosphorylated using the
following kinases: cAMP-dependent protein kinase, protein
kinase C, calcium/calmodulin-dependeent kinase, p34cdc2, or
MAPK (Erk2) (2 h, 30 °C). The buffers used for each kinase reaction
were those recommended by the kinase supplier. For phosphorylation of
the synthetic peptides, equal amounts of individual peptides (200 µg
in 400 µl of 50 mM Tris-HCl (pH 7.4)) were incubated in
MAPK buffer (containing 200 µM ATP) with 100 units of
MAPK for 2 h at 30 °C. Immunofluorescence staining was carried
out exactly as described (19).
A Ser-73 to Ala K8 mutant was generated using a TransformerTM kit (Clonetech, Palo Alto, CA). The cDNA for human K8 was used as a template as described (25). Mutation was confirmed by sequencing followed by subcloning into the pMRB101 mammalian expression vector with an hCMV promoter-directed expression. Transfection was done in baby hamster kidney cells using LipofectAMINE as recommended by the manufacturer.
The initial purpose of our study
was (i) to generate mAbs that specifically recognize phosphorylated K8
or K18 using a strategy of immunizing mice with hyperphosphorylated
keratins, and (ii) to identify the phosphorylation sites that are
recognized by such antibodies and study the biologic context of their
phosphorylation. Hyperphosphorylated keratins were purified from
human colonic cultured HT29 cells that were pretreated with the
phosphatase inhibitor okadaic acid. This antigen source should enrich
for physiologically relevant phosphorylation sites given that treatment of HT29 cells with okadaic acid results in massive hyperphosphorylation of K8/18 (19). Using this approach, a mAb termed LJ4 was generated that
preferentially immunoblotted a single species upon inducing hyperphosphorylation of keratins in cultured cells using heat stress or
okadaic acid treatment (Fig. 1B). The
increased reactivity of the antibody was not related to keratin protein
levels since an independent antibody (LJ5) that recognizes
phosphorylated and nonphosphorylated keratins (not shown) afforded
similar levels of reactivity (Fig. 1B).
Using immunoprecipitation, mAb LJ4 recognizes the K8/18/19 complex that
is also immunoprecipitated by mAb L2A1 (Fig. 1A), which in
turn recognizes the total keratin pool (18). When compared with L2A1
precipitates, LJ4 preferentially precipitates a species that migrates
slightly slower than K8 (Fig. 1A, compare lanes a-c with d-e). This slower migrating
species is similar to what we previously termed HK8 (i.e.
hyperphosphorylated K8; Ref. 11). In fact, in some experiments only
HK8-like and K18 species are noted after precipitation with the LJ4
antibody (e.g. Fig. 3D, lane a), and the
preferential binding of LJ4 with the HK8-like species is also prominent
in cells grown at 37 °C (Fig. 1A, lane d) in contrast
with a barely discernible HK8 using the L2A1 antibody (Fig. 1A,
lane a). Selectivity of the LJ4 antibody to the HK8 species was
confirmed by immunoblotting individually purified keratins from cells
grown at 37 or 42 °C (Fig. 2A, note the
intense signal with less efficiently transferred HK8). The slight
reactivity of the antibody with individually purified K8, K18, and K19
from heat-stressed cells is likely to be primarily related to slight degradation of the HK8 species during the purification process since
immunoblotting of minimally manipulated total cell lysates (e.g. Fig. 1B) shows minimal cross-reactivity.
Analysis of the phosphorylated isoforms of K8 and HK8 that are isolated
from 42 °C stressed cells showed a shift by one phospho-isoform
(i.e. phospho-isoforms of K8 and HK8 begin with
isoforms 2 and 3, respectively, Fig. 2B,
panels a-d). The assignment of the phospho-isoforms
(versus the non-phosphorylated isoform 1) is based on
metabolic labeling with 32PO4 as described in
several studies (18, 19, 23). Of note, dephosphorylation of HK8 using
alkaline phosphatase generates K8 (11, 12), thereby indicating that HK8
formation occurs via a phosphorylation event.
Identification of K8 Ser(P)-73 as the Binding Site of the LJ4 Antibody
To identify the phosphorylation site recognized by mAb LJ4, we tested several kinases for their ability to phosphorylate K8 with subsequent formation of HK8. Of several kinases tested (Fig. 3A), only MAPK resulted in substoichiometric formation of HK8 (Fig. 3A, lane f, faint band indicated by a small arrow). Since MAPK is a proline-directed kinase with preferential phosphorylation of serines adjacent to prolines (27), inspection of the K8 amino acid sequence (not shown) suggested one likely candidate region with the sequence, 67VNQSLLSPLVLE. A second candidate K8 region, 428GLTSPGL, corresponds to Ser-431 which becomes phosphorylated after epidermal growth factor stimulation but does not generate an HK8-like species (not shown).
Several peptides were synthesized that corresponded to altering the two potential phosphorylation sites Ser-70 and Ser-73 (Fig. 3B). Dot blot analysis of these peptides, before and after phosphorylation by MAPK, showed that only phosphopeptides 1 and 2 (Fig. 3B) bound with mAb LJ4, and phosphopeptide 2 showed a slightly higher LJ4 binding by ELISA (Fig. 3C). In addition, phosphopeptide 2 completely abrogates the ability of mAb LJ4 to immunoprecipitate HK8/K18, whereas phosphopeptide 1 shows partial inhibition (Fig. 3D). One possible explanation for the preferential binding of LJ4 to phosphopeptide 2 versus 1 is that in vitro phosphorylation at Ser-70 either interferes with binding of the LJ4 antibody and/or with phosphorylation at Ser-73.
Further confirmation that Ser(P)-73 is the HK8 phosphorylation site that is recognized by mAb LJ4 was obtained by generating a Ser-73 to Ala (S73A) K8 mutation. As shown in Fig. 3E, LJ4 binds to HK8 after immunoblotting of K8/18 precipitates obtained from transfected wild-type K8 and K18 but does not bind to K8/18 precipitates obtained from wild-type K18 and S73A K8 co-transfectants. In addition, mAb LJ4 reactivity is not affected upon immunoblotting of an S431A K8 mutant that is co-transfected with wild-type K18 (not shown). Taken together, the data in Figs. 1, 2, 3 indicate that K8 is phosphorylated in vivo on Ser-73 with resultant formation of HK8 and that 71LLpSPLVLE of K8 contains the epitope that is recognized by the LJ4 antibody. The possibility of constitutive K8 Ser-73 phosphorylation that is coupled to Ser-70 dephosphorylation, with subsequent mAb LJ4 reactivity, can be excluded since HK8 formation depends on a phosphorylation event (of Ser-73) (e.g. Fig. 3A), and because of the two-dimensional results of Fig. 2 and our previous data that demonstrate K8 hyperphosphorylation upon mitotic arrest and cell stress (11, 12).
Stress and Apoptosis Are Associated with Phosphorylation of K8 on Ser-73We used mAb LJ4 to examine K8/18 filament organization in
HT29 cells and in intact animals that are subjected to heat- or drug-induced stress, respectively. In the case of HT29 cells that were
cultured at 42 °C, reactivity with the LJ4 antibody was patchy in
that some cells reacted intensely with the antibody with surrounding cells showing absent staining (Fig. 4, panels
a and d). The majority of cells that bound to the
antibody manifested a reorganized dot-like staining pattern (Fig. 4,
panel a), which overlapped with the staining pattern
observed with a rabbit antibody to K8/18 (not shown). In addition, some
cells manifested a filamentous staining pattern after heat stress that
was not reorganized in any obvious fashion (not shown).
The effect of stress was also tested in mice, in the form of hepatotoxic stress that is induced after feeding mice the hepatotoxic drug griseofulvin (GF) (13). Using that model, we previously showed that GF feeding of normal mice (e.g. Balb/c) or transgenic mice that overexpress wild-type human K18 (termed TG2) resulted in liver toxicity in association with marked hyperphosphorylation of K8/18. We used this animal model to investigate if GF-induced hepatotoxicity is associated with phosphorylation of mouse K8 since the motif surrounding human K8 Ser-73 (67VNQSLLSPLVL) is very similar to Ser-79 of mouse K8 (73VNQSLLSPLKL) (Table I). As shown in Fig. 5A, GF feeding results in phosphorylation of mouse K8 as determined by immunoblotting of equivalent fractions of liver homogenates with mAb LJ4. Immunoblotting of the identical homogenates with a polyclonal antibody, which recognizes human K8/18 and also cross-reacts with mouse keratins, showed an increase in the protein level of K8/18 in some of the GF-fed mice (Fig. 5A) as described previously (28). However, any increase in K8/18 levels does not account for the observed reactivity with the LJ4 antibody after GF feeding. Confirmation that mAb LJ4 recognizes mouse K8 was obtained by immunoprecipitation, which results in co-precipitation of HK8/K18 only after GF feeding (Fig. 5B). Double immunofluorescent staining of liver sections showed absent LJ4 staining in livers obtained from control diet-fed mice which become strongly positive in a patchy pattern after GF feeding (Fig. 5C, panels e-h). In contrast, the anti-mouse K8 Troma I antibody manifests a typical filamentous pattern in all hepatocytes obtained from control diet-fed mice which becomes diffuse with scattered dots and short filaments after GF feeding (Fig. 5C, panels a-d).
|
We also examined generation of the HK8 species in cells that are induced to undergo apoptosis. This was prompted by the observation of very bright LJ4 staining of HT29 cells that was occasionally seen upon staining colcemid-treated mitotically arrested cells in association with nuclear fragmentation (not shown). The induction of apoptosis by anti-microtubule agents such as colcemid has been described previously (29). Hence, we tested K8 Ser-73 phosphorylation in association with apoptosis by treating HT29 cells with anisomycin or etoposide which are known to induce apoptosis in several tested cell lines (30, 31). As shown in Fig. 4 (panels c and f), etoposide treatment induced apoptosis in some cells, with nuclear fragmentation and a marked increase in mAb LJ4 reactivity. The increase in LJ4 reactivity is not restricted to cells that have undergone nuclear fragmentation, as clearly demonstrated after treatment of cells with anisomycin that results in intense LJ4 staining in most but not all cells (Fig. 4, panels b and e). K8 Ser-73 phosphorylation, as determined by LJ4 immunoreactivity after anisomycin and etoposide treatment, was also associated with formation of HK8 (confirmed by immunoprecipitation, not shown but a very similar pattern to that in Fig. 1) at levels that are similar to those obtained after heat stress. The filamentous staining pattern in the majority of anisomycin, and in some of etoposide-treated cells, suggests that HK8 formation is not a late event during the course of apoptosis.
HK8 Is Also Formed during Mitosis in Cultured Cells and Normal TissuesAlthough prominent levels of HK8-like species form after
mitotic arrest of HT29 cells using anti-microtubule agents or okadaic acid (12, 18), other means of obtaining mitotic cells such as
enrichment after using the DNA polymerase- inhibitor aphidicolin (32) do not result in clear detection of HK8. Therefore, we used mAb
LJ4 binding to investigate if K8 Ser-73 becomes physiologically phosphorylated during mitosis in cultured HT29 cells and in intact mouse tissues. As shown in Fig. 6A,
(a-d), the LJ4 antibody binds selectively to mitotic cells
that are obtained after aphidicolin sychronization or that are
visualized in mitotic cells that are exponentially growing after being
subcultured without the use of aphidicolin (not shown). However, for
unclear reasons, this finding was not uniform in that few clearly
mitotic cells did not bind to LJ4 (not shown). Immunoblotting of
mitotically enriched HT29 cells resulted in a significant increase in
LJ4 binding as compared with G0/G1 cells,
although the level of formed HK8 in G2/M synchronized cells
was barely visible by Coomassie staining (Fig. 6B,
right lane) as compared with G2/M-arrested cells
(12).
To address K8 Ser-73 phosphorylation in mitosis in the context of a
physiologic tissue, we asked if the LJ4 antibody can stain intestinal
crypt mitotic cells. As shown in Fig. 6C, intensely bright
staining of mitotic cells was noted in the basal crypt compartment that
contains the mitotic cells (exemplified by the metaphase cell shown in
panel h). Similar results were noted after staining mouse
small intestine (not shown). The LJ4 positive-staining mouse
colonocytes were of simple epithelial enterocytic lineage as determined
by double staining using LJ4 and rabbit anti-K8/18 antibodies (not
shown but similar to findings in the liver described below). In
addition, we examined mouse livers after partial hepatectomy as another
physiologic mitosis model. As shown in Fig.
7A, HK8 becomes detectable by immunoblotting
beginning 24 h post-hepatectomy but is not detected in pre- or
sham-hepatectomized mice. Immunofluorescence staining confirmed the
immunoblot results (Fig. 7B, panels a-c), and double
staining of the liver sections showed that the LJ4-positive cells are
simple epithelial cells (Fig. 7B, panel d). Furthermore, double nuclear/LJ4 staining (Fig. 7C) showed that the
LJ4-positive cells are indeed mitotic.
Several approaches have been successfully used to identify in vivo phosphorylation sites of IF proteins as well as hundreds of other proteins. This encompasses the use of several biochemical and analytical techniques including mass spectrometry and/or proteolysis coupled with high performance liquid chromatography (or two-dimensional peptide mapping) separation of peptides then microsequencing. Alternatively, in vitro identified phosphorylation sites that are associated with specific peptides can be matched with in vivo phosphorylated peptides. The approach utilized in this study was to (a) enhance the stoichiometry of several K8/18 phosphorylation sites by culturing cells in the presence of okadaic acid, (b) immunize mice with the purified K8/18 from these cells, and (c) select antibodies that specifically recognize phosphorylated K8 or K18 epitopes. Any generated antibodies would be predicted to recognize in vivo phosphorylation sites and could then be used as probes for the specific site and to aid in the identification of that site. Using this approach, we generated several additional antibodies to K8 and K18, including antibodies that recognize the already characterized Ser(P)-431 of K8 (14). The sensitivity and utility of these antibodies are exemplified by the discussion below on K8 Ser-73 phosphorylation during mitosis. The general utility of antiphospho-IF protein antibodies has been reviewed (23, 33).
The reverse immunological approach utilized in this study has several limitations that warrant mention. For example, the antigenicity of the generated hyperphosphorylation states and their abundance play an important role in the nature of the generated antibodies. In turn, the abundance of a particular phospho-epitope is related to the type of phosphatase inhibition used. One clear advantage of this approach is that it potentially allows for the enrichment of in vivo relevant phospho-epitopes that may otherwise have too low of a stoichiometry to be identified using routine biochemical and analytical means. To that end, enrichment of Ser(P), Thr(P), and Tyr(P) epitopes can be done using a variety of phosphatase inhibitors. One plausible variant of the above described approach is to phosphorylate in vitro an IF protein with a particular kinase, which may phosphorylate in a specific and nonspecific manner one or more in vivo relevant sites and then use the generated phosphoprotein as an immunogen to generate and screen for in vivo relevant anti-phospho-epitope antibodies.
Phosphorylation of Ser-73 during Mitosis and ApoptosisThe first observation of an HK8 species was in mitotically arrested cultured epithelial cell lines (12, 18). For example, significant and easily detectable levels of HK8 were noted upon G2/M arrest using a variety of agents including okadaic acid, nocodazole, and colcemid. However, we were unable to demonstrate a clearly detectable HK8 species in mitotically enriched cells (up to 90% G2/M cells) that were obtained after aphidicolin synchronization or by mechanical shaking (12), although a barely visible Coomassie-stained band that corresponds to HK8 is occasionally seen. Our data here clearly demonstrate that HK8 does form during mitosis, but its steady state levels are low in cultured HT29 cells. Formation of HK8 was noted within the proliferative compartment of mouse colon (Fig. 6) and small intestine (not shown), in dividing hepatocytes after partial hepatectomy (Fig. 7), and in mitotic HT29 cells (Fig. 6). Demonstration of HK8 during physiologic mitosis in tissues was made feasible using the anti-HK8 antibody that would have otherwise been missed due to its relatively low levels (i.e. few dividing cells in self-regenerating tissues).
The function of the HK8 species during mitosis and the reason(s) for its low basal level during "physiologic" mitosis (in contrast with mitotic arrest) in HT29 cells remain to be investigated. However, our results provide several potential explanations for HK8 accumulation upon mitotic arrest. First, one contributor to HK8 accumulation in G2/M-arrested HT29 cells (which include floating and adherent cells) are the generated apoptotic cells. Second, although clearly apoptotic cells (i.e. those exhibiting nuclear fragmentation) represent only a small fraction of the floater G2/M-arrested cells, most of the remaining cells have reached an irreversible state in that they are "locked" at G2/M and/or an apoptotic pathway. For example, treatment of HT29 cells with anisomycin, which induces apoptosis, is associated with a near-uniform formation of HK8 in most treated cells prior to the generation of nuclear fragments (Fig. 4). This indicates that HK8 formation is an early intermediate event along the pathway of apoptosis. Third, G2/M cell arrest using anti-microtubule agents or okadaic acid can be considered a form of toxic drug-induced stress. Therefore, one or more of these factors could explain the HK8 accumulation. Hence, formation of low levels of HK8 is normal in the life cycle of a dividing cell, but accumulation of HK8 is abnormal and signals cell stress and/or apoptosis (see below).
With regard to the function of Ser-73 phosphorylation during mitosis, several possibilities can be considered. One possibility is a role in filament organization particularly because this phosphorylation site is located within the H1 domain and is 14 amino acids away from domain 1A of the rod. Mutations within the H1 domain, in residues different than the Ser-73 K8 equivalent, have been identified in K1 (34), K5 (35), and in some cases have been attributed to abnormal filament assembly (34, 36). Although K8 Ser-73 is somewhat similar to Ser-22 of lamin C (37) in that both are phosphorylated during mitosis by a proline-directed kinase, several differences can be noted. For example, Ser-22 of lamin C is only 5 amino acids from the rod (versus 16 amino acids for K8), does not have a similar sequence to that surrounding K8 Ser-73, and does play some role in nuclear lamina reorganization (37). In contrast, phosphorylation of K8 at Ser-73 can maintain a filamentous pattern (e.g. Fig. 4b), and the complete disorganization of the keratin filament network in hepatocytes of GF-fed mice was associated with HK8 formation in only a small subset of the cells (Fig. 5C). Also, although keratins purified from heat-stressed cells (which include K18, K8, and HK8) have a higher soluble component, as compared with K8/18 isolated from non-heat-stressed cells, they do make bona fide filaments after in vitro filament assembly (11). We cannot, however, exclude a more subtle effect by K8 Ser-73 phosphorylation on filament organization. A second possibility is that K8 Ser-73 phosphorylation may positively, or negatively, regulate an interaction with another cellular element.
Phosphorylation of K8 at Ser-73 Is a Marker of Cell Stress in Simple EpitheliaOur results showed that two modalities of stress, heat in cultured HT29 cells (Figs. 1 and 4) and GF-induced hepatotoxicity in mice (Fig. 5), were associated with generation of HK8 (i.e. Ser-73 K8 phosphorylation). We define hepatotoxicity here as a form of stress since we previously showed that GF-associated hyperphosphorylation is not related to mitosis based on the lack of hyperphosphorylation of K18 Ser-52 that occurs in mice after partial hepatectomy (13). However, we cannot exclude the possibility that apoptosis may play some role in the observed generation of HK8 after GF feeding of mice. Although heat stress induces apoptosis in some HT29 cells (not shown) and in the intestine of intact animals (38), formation of HK8 occurs in the majority of cells that exhibit a condensed nuclear staining pattern (e.g. Fig. 4) that differs from that seen after anisomycin or etoposide treatment. Furthermore, heat stress is associated with induction of hsp70 in HT29 cells, which is not the case after anisomycin or etoposide treatment (not shown). In the case of heat-stressed HT29 cells, induction of HK8 occurs at a stage prior to irreversible heat-induced damage and accumulates to a high stoichiometry with stress prolongation (11). This raises the possibility of an adaptive survival-type function for this phosphorylation and indicates that it can serve as a unique marker for cell stress in simple epithelia.
Examination of the K8 sequence that surrounds Ser-73 as compared with
other type II keratin sequences shows a high degree of homology (Table
I). Our results indicate that Ser-73 is phosphorylated in
vivo, as determined by the anti-HK8 antibody reactivity with the
phosphorylated and nonphosphorylated peptides and the mutational analysis shown in Fig. 3. The LJ4 reactivity to mouse K8 (which has a
Lys in the sequence LSPLK instead of Val-76 in the human K8
sequence 71LLSPLV) (Figs. 5, 6, 7), coupled with
the lack of LJ4 reactivity with the phosphopeptide ... .
.SLLAPLV... . . but the strong reactivity with the phosphopeptide
... . ALLSPLV... . (Fig. 3), indicate that the epitope that
is recognized by the anti-HK8 LJ4 antibody encompasses the sequence
phospho-LLSPL. Inspection of type II keratin residues that correspond
to K8 Ser-73, which may serve as potential phosphorylation sites, shows
that K4, K5, and K6 have a threonine instead of a serine (Table I).
Examination of mouse skin and esophagus that express K5 and K4,
respectively, did not show cross-reactivity, possibly due to the Ser
Thr substitution of the epitope. Interestingly, K4/5 and K6 are
expressed within tissue compartments that have a regenerative capacity
(K4 and K5 in the esophagus and skin, respectively) or in the
"stress" context of skin wound healing. This is somewhat analogous
to K8 expression in potentially regenerating simple epithelial tissues
such as the intestine and liver. Therefore, one possible model that
will require testing is that the threonines in K4/5/6 that correspond
to Ser-73 of K8 may also be phosphorylated in settings that are similar
to those observed for K8.
We are very grateful to Letty Esguerra for preparing the manuscript, Kris Morrow for preparing the figures, Rosemary Fernandez for peptide synthesis, Evelyn Resurreccion for assistance with tissue sectioning and staining, Lori A. Lowthert and Dan Price for assistance with some of the experiments, and Phuoc Vo for assistance with the fusion.