(Received for publication, July 25, 1994; and in revised form, September 29, 1994)
From the
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.
Intermediate filaments (IF) ()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 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
72,000) and the
constitutively expressed hsc70 (M
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
-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,
-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.
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.
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
g, 1 h, 4 °C) and then SDS-PAGE of equivalent
fractions of the soluble and pellet fractions.
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).
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
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).
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).
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 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
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).
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 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
-ATP
S (lane5),
Mg
(lane6), and ATP
S (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
(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
(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 72,000, lower band of
doublet in Fig. 8A, lanea), whereas
cells grown at 37 °C showed a predominance of the constitutive
hsc70 (M
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.
Figure 9:
Specific binding of hsp70 to K8. Panel
A, -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.''
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.
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%.
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.
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.