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Volume 270, Number 2, Issue of January 13, 1995 pp. 915-922
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The 70-kDa Heat Shock Proteins Associate with Glandular Intermediate Filaments in an ATP-dependent Manner (*)

(Received for publication, July 25, 1994; and in revised form, September 29, 1994)

Jian Liao (§), ,  Lori A. Lowthert,  Nafisa Ghori,  M. Bishr Omary (§)

From the Palo Alto Veterans Affairs Medical Center, Palo Alto, California 94304 and the Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305-5487

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament proteins expressed preferentially in glandular epithelia. We describe the identification, by co-immunoprecipitation from normal human colonic tissues and cultured cell lines, of the 70-kDa heat shock protein (hsp) and its related heat shock cognate protein as K8/18-associated proteins (hsp/c). The association is significant but sub-stoichiometric and occurs preferentially with the soluble rather than the cytoskeletal K8/18 fractions. Heat stress increases the level of soluble K8/18 in association with an increase in hsp70 levels and an increase in the stoichiometry of K8/18-hsp70 association. Identity of the associated proteins was confirmed by microsequencing of a tryptic digest of the purified associated protein and by using anti-hsp/c70-specific antibodies. The K8/18-hsp/c70 complex can be dissociated in a Mg-ATP-dependent manner that requires ATP hydrolysis. Binding of hsp to K8/18 can be reconstituted using purified bovine hsp70 and human K8/18 immunoprecipitates that have been depleted of bound hsp/c70 and increases slightly in the presence of ATP. The reconstituted K8/18-hsp70 complex can be again released in the presence of Mg-ATP. In addition, hsp70 binds to K8/18 without having a significant effect on in vitro filament assembly when added during or after assembly. Using an overlay assay, hsp70 binds exclusively to K8 in the presence of ATP. Our results show direct association of the hsp/c70 proteins with K8/18. This interaction may serve, at least in part, to regulate the function of these two abundant protein groups.

INTRODUCTION

Intermediate filaments (IF) (^1)comprise one of the three major cytoskeletal protein groups that include microfilaments and microtubules (for reviews, see Steinert and Roop, 1988; Klymkowsky et al., 1989; Skalli and Goldman, 1991; Fuchs and Weber, 1994). IF expression is generally tissue specific with the keratin IF subfamily being preferentially expressed in epithelial cells. Tissue-specific expression within epithelial subtypes also extends to keratins, which are comprised of at least 20 members (catalogued as K1-K20) that are found in cells as heteropolymeric pairs (Moll et al., 1982, 1990). Although each epithelial cell type may express several keratins, they usually express one pair as their dominant IF network. For example, glandular ``simple'' type epithelia, as found in the gastrointestinal tract, liver and pancreas express K8 and K18 (K8/18), keratinocytes express K5/14, and esophageal epithelia express K4/13 as their major keratin pair. IF proteins are highly abundant in cells that express them, as noted for K8/18, which make up 5% of the total protein in cultured colonic cell lines (Chou et al., 1993), and for keratinocyte keratins, which account for 30% of the cellular protein (Sun et al., 1979).

A number of intermediate filament-associated proteins (IFAP) have been described that may be involved in regulating the function of IF or that may utilize binding to IF to regulate their function (Foisner and Wiche, 1991). These IFAP may interact directly or indirectly with IF and include kinases, membrane-associated proteins, and nuclear proteins. Examples of IF-kinase interactions include the association of protein kinase C-related kinases with K8/18 (Omary et al., 1992a) and vimentin (Spudich et al., 1992; Murti et al., 1992) and the association of cGMP-dependent protein kinase with vimentin (Wyatt et al., 1991; MacMillan-Crow and Lincoln, 1994). Other IFAP include ankyrin and lamin B (Georgatos et al., 1987; Djabali et al., 1991), desmoplakin (Stappenbeck et al., 1993), a 140-kDa desmosomal antigen (Cartaud et al., 1990), IFAP 300 (Skalli et al., 1994), plectin (Foisner et al., 1991), gyronemin (also called filamin) (Brown and Binder, 1992), and KAP85 (Chou et al., 1994).

Another highly abundant protein family is the heat shock protein family (HSP) with major classes including HSP25, -60, -70, -80, -90, and -110 (numbers correspond to approximate M(r) of class members) (Gething and Sambrook, 1992; Georgopoulos and Welch, 1993; Hendrick and Hartl, 1993; Becker and Craig, 1994). An important function of HSP proteins is to act as molecular chaperones, and in doing so they form complexes with a variety of cellular proteins (e.g. see Schlesinger, 1990). In mammalian cells, two cytosolic members of the HSP70 family are the heat-inducible hsp70 (M(r) 72,000) and the constitutively expressed hsc70 (M(r) 73,000) (Becker and Craig, 1994). Several indirect reports and reviews have suggested an interaction of HSP70-like proteins with IF, although most describe an association of HSP70 with microtubules (e.g. Schlesinger, 1990; Gupta, 1990). For example, 1) a ubiquitous 70-kDa protein termed beta-internexin was initially identified as a microtubule-associated protein and shown indirectly to associate with IF (Napolitano et al., 1985) and then was subsequently shown to be identical to hsp70 (Green and Liem, 1989); 2) hsp70 was shown to co-purify with microtubules in murine mastocytoma cells (Ohtsuka et al., 1986) and with retinal microtubule and IF fractions (Clark and Brown, 1986); 3) in vitro interaction of purified yeast HSP70 with vimentin and nuclear lamins in an ATP-dependent manner has been reported (Georgatos et al., 1989); 4) treatment of cultured rat brain cells with the natural product withangulatin A resulted in increased vimentin and hsp70 levels in the detergent-nonextractable fraction, although direct association was not shown (Lee et al., 1993). More recently, alpha-crystallins, which are related to the HSP25 family, were shown to co-immunoprecipitate with vimentin in bovine lens and to inhibit in vitro filament assembly of vimentin and glial fibrillary acidic protein (Nicholl and Quinlan, 1994).

During our studies of the post-translational modification of K8/18, we noted a 70-kDa band, visible by Coomassie staining, that co-immunoprecipitated with K8/18 (Chou et al., 1993). In this report, we show that this 70-kDa band corresponds to hsp/c70 and that its association with K8/18 increases with heat stress. K8/18-hsp/c70 association occurs preferentially with the soluble K8/18 fraction, with release of hsp in the presence of Mg-ATP. Furthermore, K8/18 and hsp70 binding and Mg-ATP-mediated release can be reconstituted using purified hsp70 and K8/18 immunoprecipitates that have been depleted of bound hsp/c70. In vitro binding of hsp70 to keratins using an overlay assay occurs exclusively with K8. Addition of purified hsp70 to K8/18 during or after filament assembly does not affect assembly. The potential functional significance of the K8/18-hsp70 association is discussed.

MATERIALS AND METHODS

Reagents

Purified bovine brain hsp, alpha-actinin, and ATPS were purchased from Sigma. Monoclonal antibodies (MAb) to K8/18 that recognize different K8/18 epitopes (not shown) were CK5 and 8.13 ascites (Sigma) and L2A1 (Chou and Omary, 1991), which was used as ascites or covalently coupled to protein A-agarose. Other MAb used were M2B3, which recognizes a proliferation-associated cell surface glycoprotein (Omary et al., 1992c), and I4D4 (Omary et al., 1992b), which recognizes an epithelial cell adhesion marker termed KS-1 or more recently referred to as Ep-CAM (Litvinov et al., 1994). The latter two MAb were coupled to cyanogen-activated Sepharose (Pharmacia Biotech Inc.) as recommended by the manufacturer. Antibodies to hsp70(1197) and hsp/c70 (BRM-22) were purchased from Amersham Corp. and Sigma, respectively. S protein-labeling mix (Met/Cys, 1200 Ci/mmol) and ENHANCE were from DuPont NEN.

Cell Culturing, Metabolic Labeling, and Colonic Tissues

HT29, T84, Caco2, and SK-CO-1 cell lines (human colon) were obtained from the American Type Culture Collection and were grown at 37 °C (10% CO(2)) in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 1 mM glutamine. For heat stress, cells at 50% confluency were grown at 42 °C for 16-24 h. Colonic biopsies (2-3 mm in diameter) were obtained from patients undergoing routine colonoscopy for polyp screening under a protocol approved by the Medical Committee for the Protection of Human Subjects on Research at Stanford University.

Metabolic labeling of HT29 cells with [S]Met/Cys was done by pre-incubating cells (1 h) in Met-free RPMI 1640 medium supplemented with 10% fetal calf serum dialyzed against 0.15 M NaCl, followed by addition of the S label (100 µ Ci/ml) for 1, 5, or 10 min. Cells were then immediately placed over ice, followed by removal of the labeling medium and rinsing with cold phosphate-buffered saline (PBS) containing 15 µg/ml unlabeled Met. In some cases, cells were chased for 1-20 h by incubating in normal growth medium after rinsing off the label.

Immunoprecipitation

Cells were solubilized (30-60 min, 4 °C) in 1% Nonidet P-40 in PBS containing 0.1 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 10 µM leupeptin, 10 µM pepstatin, and 5 mM EDTA (buffer A). Colonic biopsies were homogenized using a Dounce in buffer A (100 strokes) and then were allowed to solubilize with rocking for 1 h. Nonsolubilized material was pelleted (16,000 times g, 30 min, 4 °C), and the solubilized material was used for immunoprecipitation. Alternatively, HT29 cells were disrupted by nitrogen cavitation (150 p.s.i., 5 min, 4 °C) in PBS, 5 mM EDTA followed by pelleting (16,000 times g, 30 min, 4 °C) to generate the soluble (S) fraction. The insoluble fraction was solubilized in buffer A (1 h) followed by repelleting to generate the detergent-solubilized P fraction. Immunoprecipitates were then obtained using the S fraction (directly or after adding detergent to a final concentration of 1% Nonidet P-40), buffer A-solubilized cells or biopsies, or the detergent-solubilized P fraction. For this, 200-500 µl of lysate (from 2-5 times 10^6 cells or 2 biopsies) were mixed with 20 µl of agarose- or Sepharose-coupled antibody or 3 µl of ascites. The ascites-antigen complexes were then collected using protein A-Sepharose conjugated to rabbit anti-mouse antibody.

Protein Analysis

Immunoprecipitates were analyzed using SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970). Isoelectric focusing (Chou et al., 1992) and Western blotting (Towbin et al., 1979) were as described except that an enhanced chemiluminescence system (ECL) was used as recommended by the manufacturer (Amersham). Relative levels of K8/18 and hsp/c70 were determined using densitometric scanning of Coomassie stained-destained gels (LKB Ultrascan XL enhanced laser densitometer). Serial dilutions of bovine serum albumin (BSA) as a protein standard were analyzed on the same SDS-PAGE gel containing the proteins of interest.

Filament Assembly and Fast Protein Liquid Chromatography Purification of K8/18

Purified K8/18 were obtained prior to filament assembly using a two step procedure. The first step involved high salt extraction exactly as described (Achtstaetter et al., 1986; Chou et al., 1993). This generated a highly enriched K8/18, which is then solubilized in 6 M urea, 50 mM Tris-HCl, 2 mM EGTA (pH 7.5) (buffer B). Non-solubilized material was pelleted (16,000 times g, 2 min) followed by filtering of the solubilized material (0.2-µm filters) and then passage over a Mono-Q Resource column assembled in a fast protein liquid chromatography apparatus (Pharmacia). K8/18 were then eluted using a 0-0.5 M guanidine HCl gradient (in buffer B) with the K8/18 peak being eluted between 130 and 260 mM guanidine HCl. Average yield of purified keratin was 50 µg per confluent 100-mm dish of HT29 cells. The eluted keratins were dialyzed against buffer B (4 °C, 12-24 h) and then stored at 4 °C till further use.

Filament assembly was initiated by dialyzing K8/18 (150 µg/ml) against 50 mM Tris-HCl, 2 mM EGTA (pH 7.5) for 36 h with two buffer changes. Assembly was also done in the presence of bovine hsp70 or BSA. Alternatively, filaments were assembled for dialysis for 36 h and then incubated with BSA or hsp70 for 24 h at 4 °C. Filament formation was assessed by negative staining followed by electron microscopy as described (Chou et al., 1993). Analysis of the soluble K8/18 fraction after assembly in the presence or absence of hsp70 or BSA was assessed by ultracentrifugation (300,000 times g, 1 h, 4 °C) and then SDS-PAGE of equivalent fractions of the soluble and pellet fractions.

Release of hsp/c70 from K8/18 and Reconstitution of K8/18-hsp70 Binding

K8/18 immunoprecipitates were obtained from 1% Nonidet P-40 detergent lysates (of cells grown at 37 or 42 °C) in the absence or presence of 5 mM Na-ATP, Na-ATP plus MgCl(2) (Mg-ATP), Mg-AMP, Mg-ADP, Mg-GTP, ATPS, ATPS plus MgCl(2), or MgCl(2) alone. Alternatively, immunoprecipitates were isolated first and then incubated with 5 mM Mg-ATP for 15-90 min, followed by washing 2 times with PBS to remove released hsp/c70. K8/18 immunoprecipitates depleted of hsp/c70 (K8/18-hsp) were then incubated with 50 µl of PBS containing 2 µg of bovine hsp70 (60 min) in the presence or absence of 5 mM Na-ATP, Mg-ATP, or MgCl(2) followed by washing to remove unbound hsp70 and then analysis by SDS-PAGE.

Keratin-hsp70 Binding Using an Overlay Assay

A highly enriched keratin fraction was prepared by high salt extraction (Chou et al., 1993) followed by separation using preparative SDS-PAGE. Individual K8, K18, and K19 bands were visualized using copper stain (Bio-Rad), cut and electroeluted, and then concentrated using Centricon microconcentrators (Amicon). SDS-PAGE of individual K8, K18, K19, and alpha-actinin was then done, followed by transfer to two duplicate polyvinylidene fluoride membranes. One membrane was stained with Coomassie Blue, and the second was blocked with 3% BSA in PBS for 48-96 h, followed by incubation with 1 µg/ml of bovine hsp70 for 2 h in the presence of 5 mM Na-ATP in PBS containing 0.05% Tween 20 and 0.1% BSA (buffer C). After four washes with 0.05% Tween 20 in PBS (10 min/wash), the membrane was incubated with anti-hsp70 antibody in buffer C for 2 h, washed 3 times, and then incubated with peroxidase-conjugated goat anti-mouse IgG. Bound hsp was visualized using ECL.

RESULTS

Association of K8/18 with HSP70

In carrying out immunoprecipitations of K8/18 from the colonic epithelial cell line HT29, we consistently noted co-immunoprecipitation of a 70-kDa band with K8/18. This is exemplified in Fig. 1A (lanea), where a 70-kDa species is noted to co-immunoprecipitate with K8/18 using our high capacity anti-K8/18 MAb L2A1. As a control, immunoprecipitation of other antigens using well described antibodies to Ep-CAM and M2B3 antigens (Fig. 1A, lanesb and c, respectively) did not co-immunoprecipitate either K8/18 or the 70-kDa band. The Ep-CAM glycoprotein (M(r) 38,000) is abundant in HT29 cells and can be visualized by Coomassie staining (Fig. 1A, laneb), whereas the M2B3 antigen (Fig. 1A, lanec) can only be visualized after radiolabeling (Omary et al., 1992c). Purification of the 70-kDa band shown in Fig. 1A followed by trypsinization, high pressure liquid chromatography separation of the generated peptides, and microsequencing of one of the peaks provided the sequence TTPSYVAFTDTE. This sequence showed 100% homology with amino acids 37-48 of human hsp70 and hsc70 proteins (Dworniczak and Mirault, 1987). The association of K8/18 with hsp/c70 by co-immunoprecipitation was noted also in normal human colonic biopsies (Fig. 1A, lanef) and in all colonic tissue culture cell lines tested including T84, Caco2, and SK-CO-1 (not shown). The identity of a 160-kDa band that co-immunoprecipitated with K8/18 in normal colonic tissue (Fig. 1A, lanef, openarrowhead) was not investigated further. Another Coomassie-stained band that migrates between K8 and K18 (e.g.Fig. 1A, lanesa, e, and f) is usually seen with variable intensity and likely corresponds to degraded K8 or a related keratin (based on tryptic peptide mapping, not shown).

Figure 1: The 70-kDa heat shock proteins associate with keratin intermediate filaments. HT29 human colonic tissue culture cells or normal human colonic biopsies were solubilized in buffer containing 1% Nonidet P-40 (buffer A) followed by immunoprecipitation using anti-K8/18 MAb (L2A1, 8.13, and CK5) that recognize different keratin epitopes or antibodies to Ep-CAM (I4D4) and M2B3 antigen as described under ``Materials and Methods.'' Panel A, Coomassie stain of immunoprecipitates analyzed by SDS-PAGE; panel B, SDS-PAGE analysis of immunoprecipitates obtained from solubilized HT29 cells using control normal mouse ascites (lanea) or using anti-K8/18 MAb (lanesb-d). An immunoprecipitate identical to that in lanea of panelA was also blotted using anti-HSP70 antibodies (lanese and f) or using normal ascites (laneg). Openarrowheads in panelsA and B correspond to a 160-kDa nonidentified protein and to the monoclonal antibodies that bound to protein A under nonreducing conditions, respectively.


To confirm the specificity of association of hsp/c70 with K8/18, we used three MAb that recognize different K8/18 epitopes. All three MAb resulted in co-immunoprecipitation of hsp/c70 with K8/18 (Fig. 1B, lanesb-d), indicating that hsp/c70 co-immunoprecipitation is not due to cross-reaction of MAb L2A1. In addition, the identity of the 70-kDa band as hsp70 and/or hsc70 was further confirmed by blotting of K8/18 immunoprecipitates using two different MAb to HSP70 proteins (Fig. 1B, lanese and f). We were not able to adequately test if anti-hsp/c70 antibodies co-immunoprecipitate K8/18 since the two antibodies we used did not efficiently immunoprecipitate hsp/c70 (not shown).

Effect of Heat Stress on K8/18 Solubility and Association with HSP70

We studied the effect of heat stress on K8/18 association with hsp/c70 and asked how the association partitions between the soluble and cytoskeletal K8/18 pools. As shown in Fig. 2A, the amount of HSP70 associated with K8/18 increased 2.5-fold after heat stress (lanes3 and 4), and this correlated with the increased expression of hsp/c70 (lanes1 and 2). This was confirmed by blotting of K8/18 immunoprecipitates and detergent lysates obtained from normal and heat-stressed cells using anti-HSP70 antibodies (not shown). Analysis of K8/18 immunoprecipitates obtained from the S and P fractions of HT29 cells (normalized to nearly equal amounts of immunoprecipitated keratins) showed increased association of hsp/c70 with K8/18 upon heat stress in both fractions (Fig. 2A, lanes5-8). Of note, a band indicated by the dottedarrow in lanes3, 6, and 8 is co-immunoprecipitated with K8/18 from heat-stressed cells. This band, which may represent a hyperphosphorylated form of K8 that was previously observed in mitotically arrested cells (Chou and Omary, 1994), becomes more noticeable with increasing duration of heat stress (not shown).

Figure 2: Hsp/c70 associates preferentially with the soluble K8/18 fraction, with increased association, and with increased K8/18 solubility induced by heat stress. Panel A, HT29 cells (37 °C) or heat-stressed cells (42 °C, 16 h) were solubilized in Nonidet P-40 followed by analysis using SDS-PAGE of the soluble lysates after pelleting (lanes1 and 2) or K8/18 immunoprecipitates obtained from the lysates (lanes3 and 4). Alternatively, cells (37 and 42 °C) were disrupted by nitrogen cavitation followed by ultracentrifugation. The pellet (P fraction) and soluble supernatant (S fraction) were used for immunoprecipitation with MAb L2A1 (lanes5-8) as described under ``Materials and Methods.'' Panel B, equal numbers of HT29 cells (37 and 42 °C) were solubilized directly in Laemmli sample buffer (Total) or disrupted followed by ultracentrifugation and isolation of the S fraction and then SDS-PAGE analysis (lanesa-d). Equal fractions (based on cell number) of the S preparations were also used for immunoprecipitation (i.p.) of K8/18 (lanese and f). Duplicate samples were analyzed by Coomassie staining or by immunoblotting using MAb L2A1. +, indicates heat-stressed cells; -, indicates cells grown at 37 °C.


Given that HSP70 association with K8/18 occurs preferentially with the soluble K8/18 pool with increased association noted after heat stress, we asked if heat stress results in an increase in the soluble K8/18 pool. As shown in Fig. 2B, the soluble K8/18 fraction increases after heat stress when analyzed by immunoprecipitation (lanese and f) or by Western blotting of the total soluble fraction before immunoprecipitation (lanesc and d) or after immunoprecipitation (lanese and f). For the analysis shown in Fig. 2B, equal numbers of cells grown at 37 and 42 °C were used, which had nearly equal total keratin (Fig. 2B, lanesa and b).

The molar stoichiometry of hsp/c70 associated with soluble K8/18 was 1:10 and 1:25 for cells grown at 42 and 37 °C, respectively, as estimated after Coomassie staining using BSA standards (not shown). This assumes binding of hsp/c70 to either K8 or K18 and an average M(r) of 70,000 for hsp/c and 50,000 for keratin. This stoichiometry decreases nearly 3-fold when analyzing the detergent-solubilized fraction (e.g.Fig. 1B or 2A).

Binding of hsc70 and hsp70 to the Soluble and Cytoskeletal K8/18 Fractions

The 70-kDa K8/18-associated species were occasionally resolved in our gel system into two very closely migrating bands, thereby suggesting that both hsp70 and hsc70 associated with K8/18 (e.g.Fig. 2B, lanef). To confirm this, we used isoelectric focusing/SDS-PAGE and antibodies that recognized hsp70 or hsp/c70 to study K8/18 association with hsp/c70 after isolation of the complex from the soluble and cytoskeletal pellet fractions. As shown in Fig. 3, the anti-hsp70 MAb recognized a single hsp spot (panelsc and d), whereas the anti-hsp/c70 MAb recognized two spots corresponding to hsp70 and hsc70 (panelsa and b). The pI values for hsp70 and hsc70 were 5.8 and 5.5, respectively, which are similar to values previously reported for bovine hsp/c70 (Ungewickell, 1985). The hsc70 (M(r) 73,000) migrated slightly slower than the hsp70 (M(r) 72,000) species as noted by Coomassie staining (Fig. 3, panelse and f). Hence, both hsp70 and hsc70 associate with soluble and cytoskeletal K8/18.

Figure 3: Two-dimensional gel and Western blot analysis of the associated 70-kDa heat shock proteins isolated from soluble and insoluble keratin pools. HT29 cells were disrupted by nitrogen cavitation followed by centrifugation to isolate the soluble and pellet fractions. K8/18-hsp/c70 co-immunoprecipitates were obtained from the S and P fractions followed by isoelectric focusing (IEF) in the first dimension and then SDS-PAGE in the second dimension. Two-dimensional gels were then analyzed by Western blotting using MAb BRM-22 (anti-hsp/c70) and MAb 1197 (anti-hsp70). Blots used in panelsa and b were also stained to confirm the position of hsc and hsp, which are indicated by arrows, and are shown in panelse and f.


Since immunoprecipitation examines steady state levels of partitioning between the S and P fractions, we examined the K8/18-hsp/c70 association in terms of newly synthesized K8/18 and hsp/c70. As shown in Fig. 4, the majority of newly synthesized K8/18 (after a 1-min pulse label) is seen in the soluble fraction and is also associated with some newly synthesized hsp/c70. Labeling for 5 or 10 min resulted in rapid redistribution of K8/18 from the soluble to the cytoskeletal pools, with similar association with hsp/c70 (Fig. 4). As noted in Fig. 2, the steady state level for the various labeling time points, as determined by Coomassie staining of the immunoprecipitates from the S and P fractions, showed preferential association with the soluble K8/18 pool (Fig. 4B). Labeling for 10 min followed by 1-20 h chase gave results that were similar to the 10-min time point in Fig. 4A (not shown). This indicated that the K8/18-hsp/c70 association occurs not only at the early stages of biosynthesis but also subsequent to biosynthesis.

Figure 4: Hsp associates with K8/18 early during biosynthesis. HT29 cells were incubated in Met-free medium for 1 h and then labeled with [S]methionine for 1, 5, or 10 min (100 µCi/ml). After labeling, cells were immediately placed over ice and then rinsed with cold PBS containing 15 µg/ml unlabeled Met. Labeled cells were disrupted using nitrogen cavitation followed by isolation of the S and P fractions and then immunoprecipitation of K8/18-hsp/c70. Immunoprecipitates were analyzed by SDS-PAGE and Coomassie staining (panelB) followed by fluorography (panelA).


Effect of hsp70-K8/18 Binding on Filament Assembly

Given recent findings that the HSP25-related alpha-crystallins inhibited glial fibrillary acidic protein and vimentin in vitro filament assembly in an ATP-independent manner (Nicholl and Quinlan, 1994), we tested the effect of purified hsp70 on K8/18 in vitro filament assembly. This was done by adding bovine hsp70 to K8/18 proteins during assembly (i.e. before dialysis of the 6 M urea, which allows for filament assembly) or after filaments have assembled. As shown in Fig. 5, addition of bovine hsp70 to K8/18-assembled filaments for 24 h followed by ultracentrifugation resulted in binding of a small fraction of hsp70 to the cytoskeletal pellet pool in a dose-dependent manner. To exclude the possibility of protein trapping during assembly, no binding of BSA to the formed filamentous fraction was noted (Fig. 5, Pfraction, lane5). Analysis of the formed filaments by electron microscopy showed no effect of hsp70 (or BSA) on filament assembly (not shown). Similarly, inclusion of hsp70 during filament assembly resulted in binding of some of the hsp70 to the insoluble K8/18 pool in a dose-dependent fashion (Fig. 6, panelA) and did not significantly interfere with filament assembly (Fig. 6, panelB). No binding of BSA to the filamentous fraction was noted (Fig. 6, panelA, lane1). As a control, incubation of hsp70 in 6 M urea, followed by dialysis (i.e. under filament assembly conditions) and then ultracentrifugation, maintains all the hsp70 in the soluble fraction (not shown). Of note, hsp70 did not significantly alter the partitioning of K8/18 into the soluble fraction when added after (Fig. 5) or during (Fig. 6) filament assembly.

Figure 5: Assembled K8/18 filaments bind to hsp70. K8/18 was purified from HT29 cells using high salt extraction of cells to enrich for keratin proteins followed by purification using anion exchange chromatography (Mono-Q column) as described under ``Materials and Methods.'' Purified keratins (15 µg in 100 µl of 50 mM Tris-HCl (pH7.5), 2 mM EGTA, containing 6 M urea) were dialyzed to remove the urea and allow filament assembly. Assembled filaments were incubated with the indicated amount of bovine brain hsp70 or BSA as a control protein (24 h, 22 °C), followed by centrifugation to isolate the soluble (S) and insoluble (P) fractions (300,000 times g, 30 min). Equivalent fractions were then analyzed by SDS-PAGE.


Figure 6: Hsp70 does not significantly alter solubility or in vitro assembly of keratin intermediate filaments. K8/18 were co-purified by ion exchange chromatography and then used for in vitro assembly (30 µg in 200 µl) alone or in the presence of the indicated amounts of BSA or bovine brain hsp70 in 6 M urea, 50 mM Tris-HCl (pH 7.5), 2 mM EGTA. Filament assembly was initiated by dialysis against 50 mM Tris-HCl, 2 mM EGTA (pH 7.5) for 36 h with buffer change every 12 h. Dialyzed samples were analyzed by electron microscopy (panelB) or were pelleted (300,000 times g, 30 min) followed by SDS-PAGE analysis of equivalent portions of the S and P fractions (panelA). Filament assembly was similar for all the treatments and is represented by the two micrographs shown in panelB. Smallarrow shows hsp70 particles, and bar = 60 nm. Similar hsp70 particles were noted by electron microscopy if hsp70 in the 6 M urea containing buffer, in the absence of K8/18, was dialyzed under assembly conditions (not shown).


Effect of ATP on K8/18-hsp/c70 Binding and Reconstitution of hsp70-K8/18 Binding and Dissociation in Vitro

Given that the association of heat shock proteins with a number of other proteins is ATP dependent (e.g. Rothman and Schmid, 1986; Palleros et al., 1991), we tested if the interaction of hsp/c70 with K8/18 is also ATP dependent. This was carried out by adding ATP or other nucleotide derivatives to 1) detergent lysates followed by immunoprecipitation or 2) immunoprecipitates followed by washing to remove released material. As shown in Fig. 7A, addition of Mg-ATP (lane4) to detergent lysates followed by immunoprecipitation of K8/18 resulted in the release of hsp/c70 from the K8/18-hsp/c70 complex, whereas addition of non-hydrolyzable Mg-ATPS (Fig. 7A, lane5) or Na-ATP (Fig. 7A, lane3) had no effect. This suggests that ATP hydrolysis is important for the release of hsp/c70 from the complex. Similar results were obtained when immunoprecipitates of K8/18 instead of cellular lysates were used, as exemplified by the release of hsp/c70 from K8/18 (compare Fig. 7B, lanes1 and 2). Furthermore, analysis of K8/18 immunoprecipitates in the presence of individual or combinations of Na-ATP/MgCl(2) gave parallel results to those in Fig. 7A (not shown). In addition, Mg-AMP and Mg-ADP did not result in release of the K8/18-hsp70 complex, whereas Mg-GTP gave similar results to Mg-ATP (not shown).

Figure 7: Hsp association with K8/18 is ATP dependent and can be reconstituted in vitro. Panel A, a soluble fraction of HT29 cells was obtained by cell disruption followed by ultracentrifugation (300,000 times g, 30 min). Immunoprecipitation from the soluble fraction was carried out using normal ascites coupled to agarose (lane1) or with MAb L2A1 coupled to agarose (lanes2-7) in the presence of 5 mM of Na-ATP (lane3), Mg-ATP (lane4), Mg-ATPS (lane5), Mg (lane6), and ATPS (lane7). Panel B, Immunoprecipitates of K8/18 were obtained from HT29 cells grown at 37 °C (lane1) and then treated with Mg-ATP (lane2). K8/18 immunoprecipitates depleted of hsp/c70 (i.e. identical to those in lane2) were then incubated with 2 µg of bovine hsp70 (in a total volume of 50 µl) in the presence or absence of Na-ATP and MgCl(2) (5 mM) as indicated. After 60 min, samples were washed to remove unbound hsp70 and then analyzed using SDS-PAGE followed by Coomassie staining.


We also tested the reconstitution of hsp70 binding to K8/18 immunoprecipitates that were depleted of bound co-immunoprecipitated hsp/c70 (K8/18-hsp, as, for example, the material shown in Fig. 7B, lane2). As indicated in Fig. 7B (lane3), addition of hsp70 to K8/18-hsp immunoprecipitates resulted in binding of hsp70 to K8/18, which increased somewhat in the presence of ATP but not Mg-ATP (lane5) or MgCl(2) (lane6). This suggests that ATP stabilizes the binding of hsp70 to K8/18. Persistent binding of hsp70 to K8/18-hsp in the presence of hsp70 and Mg-ATP (Fig. 7B, lane5) may seem contradictory to the observed release of hsp70 from K8/18 immunoprecipitates (Fig. 7B, lane2). However, this persistent binding is likely related to the excess hsp70 in the reconstituted binding case, which drives the equilibrium toward binding rather than release.

To complete the cycle of K8/18-hsp/c70 complex release and reconstitution, we asked if the reconstituted K8/18-hsp70 (after washing off excess unbound hsp70) can be dissociated in a manner similar to the K8/18-hsp/c70 complex isolated from solubilized or disrupted cells. As shown in Fig. 8, the cycle of K8/18-hsp/c70 association by co-immunoprecipitation (lanesa), followed by release in the presence of Mg-ATP (lanesb), followed by reconstitution (lanesc), can be followed by hsp70 release again in the presence of Mg-ATP (lanesd-f). As expected, cells grown at 42 °C showed a predominance of the heat stress-induced hsp70 (M(r) 72,000, lower band of doublet in Fig. 8A, lanea), whereas cells grown at 37 °C showed a predominance of the constitutive hsc70 (M(r) 73,000 upper band of doublet in Fig. 8B, lanea). Of note, K8/18 immunoprecipitates isolated from heat-stressed cells (panelA) showed complete release of bound bovine hsp70 within 15 min, whereas release from K8/18 immunoprecipitates isolated from cells grown at 37 °C (panelB) showed partial release in a time-dependent fashion. This difference in the complete release of bovine hsp70 is highly reproducible (n = 6) and may be related to the effect of altered phosphorylation of K8/18 in heat-stressed cells (not shown) on its conformation or to the use of bovine hsp70 with human K8/18.

Figure 8: Release of reconstituted K8/18-hsp70. Immunoprecipitates of K8/18 were obtained from cells grown at 37 °C or after heat stress (42 °C, 16 h) (lanesa). The co-immunoprecipitated hsp/c70 was released from K8/18 by treatment with Mg-ATP (lanesb) followed by washing and reconstitution of the binding by incubating with bovine hsp70 (lanesc). After washing to remove unbound hsp70, the reconstituted K8/18-hsp was incubated with Mg-ATP (5 mM) for the indicated times. Samples were then washed to remove released hsp70 and then analyzed by SDS-PAGE.


Preferential in Vitro Binding of hsp70 to K8

Since K8 and K18 form heteropolymers (Steinert and Roop, 1988) and since immunoprecipitates of K8/18 also bring down K19, we asked to which of the immunoprecipitated keratins does hsp70 bind. For this, we used an overlay binding assay whereby bovine hsp70 was incubated with individually purified K8, K18, and K19 as well as alpha-actinin as a control cytoplasmic protein. As shown in Fig. 9B, hsp70 binds primarily to K8 without any significant binding to K19, K18, or alpha-actinin. Nearly equal amounts of the proteins were loaded and transferred to the blot as shown in a duplicate blot in Fig. 9A.

Figure 9: Specific binding of hsp70 to K8. Panel A, alpha-actinin (Sigma) and individually purified K8, K18, and K19 (using SDS-PAGE and then electroelution as described under ``Materials and Methods'') were transferred to a polyvinylidene difluoride membrane and then stained using Coomassie Blue. Panel B, an identical blot to that shown in panelA was blocked with BSA and then incubated with bovine hsp. After washing, the blot was incubated with anti-hsp70 antibody followed by visualization of bound hsp70 as described under ``Materials and Methods.''


DISCUSSION

The results reported in this study support the following conclusions. 1) hsp70 and hsc70 associate with K8/18 as determined by co-immunoprecipitation from normal colonic cells and cell lines, reconstitution of binding in vitro, and binding to formed filaments in vitro during or after filament assembly. 2) In isolated cell fractions, hsp/c70 bind preferentially to the soluble fraction of K8/18 with increased binding to both soluble and cytoskeletal K8/18 after heat stress. The increased binding of hsp70 to K8/18 after heat stress is also associated with an increase in the soluble fraction of K8/18. 3) Binding of purified hsp70 to K8/18 is partially facilitated by ATP, and release of the hsp/c70-K8/18 complex in detergent cell lysates or in isolated immunoprecipitates requires Mg-ATP and ATP hydrolysis. 4) Using an overlay assay, K8 shows specific and preferential binding to hsp70 in the presence of ATP as compared with K18 and K19. 5) Binding of hsp70 to K8/18 does not significantly affect filament assembly in vitro.

Specificity of hsp/c70 Binding to K8/18

Several findings in this study support a specific hsp/c70 and K8/18 interaction that is likely to play physiologic relevance and not simply represent an in vitro phenomenon. First, binding is regulated by ATP, which argues for specificity. Second, binding is noted by co-immunoprecipitation after mild detergent solubilization (i.e. non-ionic detergent as in Fig. 1) and is similarly noted after cell disruption and then isolation of the soluble fraction in aqueous buffers in the absence of any detergents (not shown). Third, binding of hsp70 to keratins in an overlay assay suggests specific binding to K8 without binding to K18, K19, or alpha-actinin (Fig. 9). However, this overlay binding assay utilizes SDS-denatured keratin substrates for binding to hsp70, and specificity of hsp70 binding to K8 under these conditions may not reflect an in vivo situation. It remains to be determined how general the binding is of hsp/c70 to keratins and other intermediate filaments and if binding of hsp70 to K8/18 is dependent on a linear sequence of K8 or whether it is dependent on a conformation of K8/18 tetramers or polymers. Furthermore, although our data suggest a direct interaction of hsp/c70 with K8/18, we cannot exclude the possibility that this interaction may be mediated by one or more additional proteins that form a large complex. To that end, hsp/c70 is the major K8/18 co-immunoprecipitated species as determined by Coomassie staining (e.g.Fig. 1A, lanea). Therefore, if other proteins are involved in such a putative complex, they would have to be significantly substoichiometric relative to hsp/70 in the K8/18-hsp/c70 complex.

The interaction of hsp/c70 with a number of cellular proteins may occur in an ATP-dependent or -independent manner. For example, hsp/c70 interacts with clathrin-coated vesicles (Schlossman et al., 1984; Ungewickell, 1985), cap32/34 actin-binding proteins (Haus et al., 1993), and p53 oncogene (Clarke et al., 1988) in an ATP-dependent manner in contrast with the ATP-independent association with topoisomerase I (Ciavarra et al., 1994). In the case of hsp70 interaction with K8/18, ATP appears to facilitate the in vitro binding with release of the complex being mediated by ATP hydrolysis.

The association of hsp70 with K8/18 is substoichiometric but is significant. We estimate that the overall lower limit of stoichiometry of association is 1 molecule of hsp/c70 to 75 molecules of K8 (assuming that binding occurs preferentially with K8 as suggested by the overlay assay in Fig. 9). This ratio is nearly tripled for the S fraction in cells grown at 37 °C and is a further 2.5-fold higher in heat-stressed cells to give a ratio of 1:10 (hsp/c70:soluble keratin). In HT29 cells, we previously showed that nearly 5% of the total cellular protein is K8/18, and 5% of K8/18 is soluble (Chou et al., 1993). If the co-immunoprecipitation data reflect steady-state K8/18-hsp/c70 association in cells, and assuming that hsp/c70 make up 0.1-1% of the total cellular protein (Chappell et al., 1986; Pelham, 1986), then we estimate that 7-67% of hsp/c70 is bound to K8/18. The predominant cytosolic localization of hsp/c70 and the lack of immunofluorescent co-localization of hsp70 with cytoskeletal elements (not shown; Ungewickell, 1985) suggest that a significant fraction of hsp/c70 is not bound to K8/18. This suggests that the percent of total hsp/c70 that is associated with K8/18 is likely to be closer to 7% than to 67%.

Regulation of hsp/c70-K8/18 Association

The ability to release bound hsp/c70 from K8/18 and then reconstitute the binding and release in the presence of Mg-ATP suggests that the interaction of hsp/c70 with K8/18 is highly regulated. Potential regulators to consider in the interaction of hsp/c70 with K8/18 include cations, ATP/GTP, post-translational modifications such as phosphorylation, and other proteins such as co-chaperonins. For example, Mg-ATP and Mg-GTP were important for release of the complex, but nonhydrolyzable Mg-ATPS was ineffective. Although potassium-ATP was recently reported to be important for the dissociation of hsp70 from its denatured substrates (Palleros et al., 1993), potassium-ATP did not result in any K8/18-hsp/c70 disassociation (not shown). This may reflect the native rather than the denatured state K8/18 in our experiments.

The potential role of a co-chaperonin-like protein or a post-translational modification as regulators of K8/18-hsp/c70 interaction remains to be investigated. With regard to a potential post-translational modification role, we find that K8/18 in heat-stressed cells are hyperphosphorylated (not shown), which may account for the increased hsp/c70 association with K8/18 in heat-stressed cells. Alternatively, the heat stress-induced increase in K8/18-hsp/c70 association may be simply related to increased hsp70 levels. Other potential regulators may be involved in K8/18-hsp/c70 interaction. For example, yeast Hsp70 interaction with unfolded protein substrates was found to be regulated by a homolog of bacterial dnaJp, termed YDJ1 (Cyr et al., 1992). To that end, several proteins that are not easily detectable by Coomassie staining (e.g. KAP85) (Chou et al., 1994) also co-immunoprecipitate with K8/18. The role that these proteins play, if any, in K8/18-hsp/c70 interaction is not known.

Potential Functions to Consider for the Interaction of hsp/c70 with K8/18

Work from a number of laboratories has accumulated evidence for a number of functions for hsp/c70. These functions include uncoating of clathrin-coated vesicles (Rothman and Schmid, 1986), interaction with newly synthesized proteins and protein folding (Beckmann et al., 1990; Gething and Sambrook, 1992), targeting to lysosomes (Dice, 1990), nuclei (Imamoto et al., 1992; Shi and Thomas, 1992), and peroxisomes (Walton et al., 1994), and regulation of DNA binding of p53 (Hupp et al., 1992). It is tempting to speculate that the interaction of two highly abundant proteins (such as hsp/c70 and K8/18) may be a mechanism that cells can use to have the two protein groups regulate each other. This hypothesis becomes attractive if our co-immunoprecipitation data reflect physiologic and steady-state association, coupled with the noted relatively significant stoichiometry of association. For example, release of hsp/c70 that is bound to K8/18 could then allow the chaperonin (or keratin) to interact with one or more of its potential substrates. The identified K8/18-hsp/c70 association should allow testing if it plays a role in any of the above mentioned heat shock protein functions. Other potential functional roles to consider for K8/18-hsp/c70 association are analogous to the participation of non-hsp70 molecular chaperones in folding of beta-actin (Gao et al., 1992) and alpha- and beta- tubulin (Gao et al., 1993) and the ATP-independent association of alpha-crystallin with vimentin with subsequent sequestration of cytoskeletal vimentin into a soluble pool (Nicholl and Quinlan, 1994). Despite a 2-3-fold increase in the association of hsp/c70 with soluble versus cytoskeletal K8/18 and that the increased solubility of K8/18 in association with heat stress is concordant with increased levels of hsp/c70, we did not find a significant effect of hsp70 on K8/18 solubility or assembly ( Fig. 5and Fig. 6). Nonetheless, a role for hsp/c70 in the stabilization or destabilization of keratin oligomers is possible.

Finally, it is important to emphasize that hsp/c70 interacts with many if not hundreds of proteins in a transient or nontransient fashion (Schlesinger, 1990; Beckmann et al., 1990). However, the interaction of hsp/c70 with K8/18 has significance from a different light if the abundance of keratins, the relative high stoichiometry of association, and the ATP dependence are considered.

FOOTNOTES

*
This work was supported by a Veterans Affairs Merit Award, National Institute on Alcohol Abuse and Alcoholism Grant AA0947A-01, the PEW Scholars Program, and Digestive Disease Center Grant DK38707. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Please address reprint requests to Dr. Liao and other correspondence to Dr. Omary at the following address: Stanford University School of Medicine, Lab Surge Bldg., Rm. P304, Stanford, CA 94305-5487.

(^1)
The abbreviations used are: IF, intermediate filaments; hsc, heat shock cognate protein; hsp, heat shock protein; HSP, heat shock protein family; MAb, monoclonal antibody; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; S and P fractions, soluble and pellet fractions; IFAP, intermediate filament-associated proteins; BSA, bovine serum albumin; ATPS, adenosine 5`-O-(thiotriphosphate).

ACKNOWLEDGEMENTS

We are grateful to Dr. James Kenny (Protein and Nucleic Acid Facility, Stanford University) for performing the amino acid microsequencing and to Linda Jacob and Sally Morefield for preparing the manuscript.

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