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J. Biol. Chem., Vol. 275, Issue 44, 34521-34527, November 3, 2000

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Identification of Mrj, a DnaJ/Hsp40 Family Protein, as a Keratin 8/18 Filament Regulatory Protein^*

Ichiro Izawa, Miwako Nishizawa, Kazuhiro Ohtakara^§, Kenzo Ohtsuka^¶, Hiroyasu Inada, and Masaki Inagaki

From the  Division of Biochemistry and the ^¶ Cell Stress Biology Research Group, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464-8681, Japan and the ^§ Department of Neurosurgery, Mie University School of Medicine, Edobashi, Tsu, Mie 514-8507, Japan

Received for publication, April 24, 2000, and in revised form, July 19, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intermediate filaments (IFs)¹ are major components of the cytoskeleton and nuclear envelope in most types of eukaryotic cells (1-6). Although structural components of other major cytoskeletal proteins (actin and tubulin) are highly conserved in different cell types, the constituent proteins of IFs show intriguing molecular diversities and are expressed in tissue-specific programs, which makes them ideal molecular markers for the differentiation state in developmental biology and pathology (7). Cytoplasmic IFs are usually organized into 10-nm diameter fibers that are prevalent in the perinuclear region, where they appear to be attached to the outer nuclear envelope membrane or to nuclear pore complexes (4). Continuous with this perinuclear array, IFs extend radially through the cytoplasm, eventually forming a close association with the plasma membrane. The intracellular IF networks were thought to be relatively stable in comparison with other cytoskeletal components such as actin filaments and microtubules; however, IFs have dynamic properties (8, 9). Accumulating data strongly suggest that assembly and disassembly of IF organization are regulated by the site-specific phosphorylation state of IFs (9-11), which is determined by kinase/phosphatase equilibria (11-14).

The keratin subfamily, which is preferentially expressed in epithelial cells, has over 20 members (keratins 1-20) that form obligate noncovalent heteropolymers of at least one type I keratin (keratins 9-20) and one type II keratin (keratins 1-8) (15). In epithelial cells, keratin filaments are typically organized in a cytoplasmic reticular network of anastomosing filament bundles involving their noncovalent linkage at the surface of the nucleus and at cell adhesion complexes. This adhesion machinery consists of desmosomes, which mediate adhesion between cells, and hemidesmosomes, which mediate the adhesion of epithelial cells to the underlying basal lamina. One role that has been ascribed to various keratin filament networks of stratified squamous epithelia is to impart mechanical integrity to cells, without which the cells become fragile and prone to rupture (16). Disruption of the keratin IF network in epidermal keratinocytes via the targeted expression of dominant-negative keratin mutants (17-19) or the introduction of null mutations (20) results in lysis of the targeted cell population whenever the skin of such mice is subjected to trivial mechanical trauma. Mutations in keratin genes, weakening the structural framework of cells, increase the risk of cell rupture and cause a variety of human skin disorders (6, 21).

Keratins 8 and 18 (K8/18) are the major components of the IFs of simple or single-layered epithelia, as found in the gastrointestinal tract, liver, and exocrine pancreas, from which many carcinomas arise. Gene targeting techniques have been used to elucidate the function of K8/18. K8 knockout mice in one strain died around day 12 from undetermined tissue damage (22), whereas in a different strain, they survived to adulthood, but colorectal hyperplasia and inflammation were present (23). K18 null mice were fertile and had a normal life span, whereas old K18 null mice developed a distinct liver pathology with abnormal hepatocytes containing K8-positive aggregates that resembled the Mallory bodies seen in human livers with alcoholic hepatitis (24). Together with the report describing a mutation in the K18 gene in a patient with cryptogenic cirrhosis (25), additional work has indicated that K18 mutations may possibly cause or result in a predisposition to liver disease (26).

Although the molecular basis for functions of K8/18 are unknown, dissection of interactions of K8/18 with associated proteins has provided clues to the physiological roles of K8/18. Several K8/18-associated proteins have been reported, including a protein kinase C-related kinase (27), an 85-kDa membrane-associated protein (28), the stress-induced and constitutively expressed heat shock protein Hsp/c70 (29, 30), the glucose-regulated protein Grp78 (31), and members of the 14-3-3 protein family (32, 33). In the present work, we screened a yeast two-hybrid library using K18 as a bait to search for new K18-associated proteins. Among the positive clones, we identified two identical ones encoding the C-terminal portion of human Mrj, a DnaJ/Hsp40 family protein. We report evidence for the in vivo association of Mrj with K18 and suggest a possible role of the Mrj-Hsp/c70 chaperone in the regulation of K8/18 filament organization.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Two-hybrid Screening-- The pGEX-2TH-K18 and -K8 plasmids were kindly provided by Dr. H. Eto (Massachusetts General Hospital, Boston, MA). The pET8c-K5 and -K14 plasmids were kindly provided by Dr. E. Fuchs (University of Chicago, Chicago, IL). DNA encoding full-length K18 was cloned into the yeast Gal4 DNA-binding domain vector pYTH9. The resulting plasmid, pYTH9-K18, was used in the two-hybrid screen of the human liver cDNA library fused to the pACT2 vector (CLONTECH, Palo Alto, CA) following the Matchmaker Two-hybrid System protocol (CLONTECH). Positive clones were screened for their potential to grow on selective medium containing 25 mM 3-aminotriazole and for the expression of -galactosidase. Yeast DNA was recovered and transformed into Escherichia coli. Plasmids containing cDNA clones were identified by restriction mapping and further characterized by DNA sequencing. Subsequent two-hybrid interaction analyses were carried out by cotransformation of plasmids containing the Gal4 DNA-binding (pGBT9) and activation (pACT2) domains into Saccharomyces cerevisiae strain Y190.

Cloning of Full-length Mrj and DNA Constructs-- Standard DNA protocols were used (34). Full-length human Mrj cDNA was amplified using human brain Marathon-Ready cDNA (CLONTECH) and PyroBest polymerase (Takara, Tokyo, Japan) with a set of primers designed according to the human Mrj cDNA sequence deposited in the GenBank^TM/EBI Data Bank (accession number AB014888). Various Mrj deletion mutants, including Mrj-N1(aa 1-100), Mrj-N2 (aa 1-146), and Mrj-C (aa 99-242), were also amplified with appropriate sets of primers and then cloned into vectors. A series of truncations of K18 were constructed in the pYTH9 plasmid by enzyme digestion and using polymerase chain reaction techniques. Yeast two-hybrid vectors encoding human cytosolic Hsc70 was constructed, using polymerase chain reaction techniques, from the pHSC7 plasmid, which was kindly provided by Dr. N. Imamoto (Osaka University, Osaka, Japan). Mouse DnaJ/Hsp40 family cDNAs, encoding Hsp40, mDj6, mDj7, mDj8, and mDj11 (35), were also subcloned into yeast two-hybrid vectors. Sequences of these constructs were verified by DNA sequencing.

Purification of Recombinant Proteins-- Recombinant full-length Mrj protein (aa 1-242) was expressed as a His₆-tagged protein using pQE30 vectors (QIAGEN K. K., Tokyo). Expression and purification of the His-tagged Mrj protein were done according to the manufacturer's protocol (QIAGEN). Full-length Mrj was also expressed as a glutathione S-transferase (GST) fusion protein in E. coli and purified on glutathione-agarose beads essentially as described (36). Recombinant human K8 and K18 expressed in E. coli using pET3a vectors were prepared as described (12). Recombinant mouse vimentin and human desmin were purified as described (37, 38).

Filament Assembly and Co-sedimentation Assay-- IF assembly in vitro was done as described (10, 12). Briefly, purified K8 and K18 were dissolved at a 1:1 (mol/mol) ratio in 10 mM Tris-HCl (pH 8.8) containing 50 mM 2-mercaptoethanol, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 7 M urea. The mixture in 7 M urea solution was dialyzed against 10 mM Tris-HCl (pH 8.8) containing 50 mM 2-mercaptoethanol, 2 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride for 24 h at 4 °C. These heterotypic complexes of K8 and K18 (0.5 mg/ml) were incubated, with or without GST or GST-Mrj, in 20 mM imidazole (pH 7.0) containing 1 mM MgCl₂ at 25 °C for 1 h. Purified vimentin or desmin was assembled in the presence or absence of GST-Mrj in 25 mM Tris-HCl (pH 7.5) containing 50 mM NaCl at 25 °C for 1 h. The reassembled complexes were subjected to centrifugation at 15,000 rpm for 30 min. The supernatants and precipitates were then analyzed by SDS-polyacrylamide gel electrophoresis.

Cell Culture-- HeLa cells and human bladder carcinoma T24 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin in an air and 5% CO₂ atmosphere with constant humidity.

Generation of Anti-Mrj Antibody and Immunoblotting-- Anti-Mrj antibody was produced in rabbits injected with recombinant His₆-tagged Mrj protein and affinity-purified with recombinant GST-Mrj fusion protein. HeLa and T24 cells were lysed with SDS sample buffer, sonicated, and boiled. Whole lysates of ~2 × 10⁴ cells were then loaded onto the lanes, resolved by SDS-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride membrane (Atto, Tokyo). The blots were incubated with anti-Mrj antibody and horseradish peroxidase-conjugated second antibody, and immunoreactive bands were visualized using chemiluminescence detection reagents (Renaissance, PerkinElmer Life Sciences). To confirm specificity of the anti-Mrj antibody, it was pre-absorbed by recombinant His-tagged Mrj protein as a control. Anti-K18 (CY-90, Sigma), anti-Hsp/c70 (BRM-22, Sigma), and anti-GST (B-14, Santa Cruz Biotechnology, Santa, Cruz, CA) monoclonal antibodies were also used as primary antibodies for immunoblotting.

Immunoprecipitation-- Cells were lysed on ice for 20 min in lysis buffer consisting of 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 10 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 1 mM Na₃VO₄, and 10 mM NaF. Lysates were clarified by centrifugation at 15,000 rpm for 30 min. Endogenous Mrj was immunoprecipitated from cell lysates with anti-Mrj antibody or control rabbit IgG and protein A-agarose beads and washed four times with lysis buffer. Immunoprecipitates were analyzed by immunoblotting with primary antibodies and horseradish peroxidase-conjugated secondary antibodies or protein A.

Immunofluorescence-- Cells grown on 13-mm coverslips were fixed by incubation for 10 min in 50% methanol and 50% acetone (v/v) at 20 °C. Methanol/acetone-fixed cells were washed three times with phosphate-buffered saline prior to blocking with 10% (v/v) goat serum in phosphate-buffered saline. For double immunostaining with anti-Mrj and anti-K18 (CY-90) monoclonal antibodies, cells were incubated with anti-Mrj antibody diluted 1:20, followed by Alexa 488-labeled anti-rabbit antibody (Molecular Probes, Inc., Eugene, OR). Next, the cells were incubated with anti-K18 antibody (1:200), followed by FluoroLink Cy3-linked anti-mouse antibody (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). To visualize a Myc tag, actin, or tubulin, cells were reacted with anti-Myc (9E10), anti-actin (C4, Chemicon International, Inc., Temecula, CA), or anti-tubulin (Sigma) monoclonal antibody, respectively, followed by Alexa 488-labeled anti-mouse antibody (Molecular Probes, Inc.) or FluoroLink Cy3-linked anti-mouse antibody. The coverslips were examined on an Olympus LSM-GB200 microscope.

Mammalian Expression Vectors and Transfection-- cDNAs encoding full-length Mrj (Mrj-F) and deletion mutants, including Mrj-N2 (aa 1-146) and Mrj-C (aa 99-242), were cloned into the mammalian expression vector pRK5-Myc for expression of Myc epitope-tagged proteins. HeLa cells were seeded at a density of 2 × 10⁴ cells/ml onto 13-mm glass coverslips in 6-well dishes the day before lipofection with 1 µg of each plasmid using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocols. Sixteen hours after lipofection, the cells were fixed for immunofluorescence studies.

Microinjection-- The affinity-purified rabbit anti-Mrj polyclonal antibody and chromatographically purified normal rabbit IgG (Sigma) were dialyzed against a buffer containing 5 mM sodium phosphate (pH 7.4) and 100 mM KCl and then concentrated to ~5 mg/ml by ultrafiltration (Centricon, Millipore Corp., Bedford, MA). Either anti-Mrj antibody or normal rabbit IgG was injected into HeLa cells using an Eppendorf microinjection system. Approximately 1 h after the injection, the cells were fixed and subjected to immunofluorescence study. The cells were stained with Alexa 488-labeled anti-rabbit antibody to detect the injected anti-Mrj antibody and cells, followed by staining for K18, actin, or tubulin.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Mrj as a K18-interacting Protein-- To investigate the function of K8/18, we screened a human liver cDNA library using the yeast two-hybrid technique and full-length K18. We obtained 73 positive clones and classified the clones by restriction analysis and nucleotide sequencing. They contained 25 independent clones of fibrinogen-, one clone of K8, and one clone of 14-3-3 protein tau. A BLAST search suggested that two independent clones encoded the C-terminal region (aa 99-242) of the human Mrj protein, a recently cloned DnaJ/Hsp40 family protein (39, 40). As DnaJ/Hsp40 family proteins interact with the Hsp70 class of chaperones, and Hsp/c70 was reported to associate with K8/18 filaments (29, 30), we further analyzed the Mrj clones. The full-length Mrj cDNA was amplified by polymerase chain reaction using a set of primers designed according to the human Mrj sequence deposited in the GenBank^TM/EBI Data Bank (accession number AB014888). The human Mrj cDNA encodes a 242-amino acid protein, and the N-terminal half of the Mrj protein contains the J domain (aa 1-74) and a glycine/phenylalanine-rich domain (aa 75-119), which are present in E. coli DnaJ (Fig. 1A). To confirm that full-length Mrj interacts with K18 and to determine if Mrj can bind to K8, epidermal K5 and K14, or type III IF proteins, we examined the interaction of full-length Mrj or the C-terminal region of Mrj with K5, K8, K14, K18, and type III IF proteins, including vimentin, glial fibrillary acidic protein (GFAP), and desmin, in the two-hybrid system (Fig. 1B). The C-terminal region of Mrj (Mrj-C, original two-hybrid fragment) strongly interacted with K18, but did not interact with K5, K8, K14, vimentin, GFAP, or desmin. In the same fashion, full-length Mrj (Mrj-F) specifically interacted with K18, but did not bind to K5, K8, K14, vimentin, GFAP, or desmin. We next asked whether other DnaJ/Hsp40 family proteins could bind to K8, K18, or other IF proteins using two-hybrid methods. Expression vectors harboring various mouse DnaJ/Hsp40 family cDNAs, including Hsp40, mDj6, mDj7, mDj8, and mDj11 (35), fused to the Gal4 activation domain were cotransformed into yeast Y190 cells with expression vectors encoding various IF cDNAs fused to the Gal4 DNA-binding domain. Among the interactions between DnaJ/Hsp40 family proteins and K8 or K18 that we tested in this study, only the Mrj-K18 interaction was strongly positive (Fig. 1C). In addition, none of these DnaJ/Hsp40 family proteins interacted with other IF proteins, including K5, K14, vimentin, GFAP, and desmin (data not shown). These results suggest that the interaction of Mrj with K18 is highly specific, although it is still possible that other untested DnaJ/Hsp40 family proteins can specifically bind to some IF proteins. We then analyzed the region of K18 containing the Mrj interaction site. For this, the binding between a series of truncations of K18 and Mrj-F or Mrj-C was determined using the two-hybrid system (Fig. 1D). The coil II region of K18 specifically interacted with both Mrj-F and Mrj-C.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Identification of Mrj as a K18-interacting protein. A, domain organization of the human Mrj protein. The J domain (aa 1-74) and the glycine/phenylalanine-rich domain (G/F; aa 75-119) are indicated. The position of the original clone encoding the C-terminal region of Mrj (Mrj-C) is also indicated. Numbers refer to amino acid positions. B, interactions of Mrj-C or Mrj-F with K18 or other IFs. Y190 cells cotransformed with various pYTH9-IFs and pACT2-Mrj-F or pACT2-Mrj-C were selected in Trp/Leu-free medium and subjected to -galactosidase filter assay. The plus signs represent the relative rates at which the transformed yeast colonies turned blue after incubation at 30 °C on filters: +++, <3 h; ++, 3-8 h; +, >8 h. The minus signs represent colonies remaining white at 24 h. Yeast cells cotransformed with pYTH9-K18 and pACT2-K8, which started to turn blue in <3 h (designated +++) during the filter assay, served as a positive control. Vim., vimentin; Des., desmin. C, interactions of various DnaJ/Hsp40 family proteins with K8 or K18. Expression vectors harboring various mouse DnaJ/Hsp40 family cDNAs, including Hsp40, mDj6, mDj7, mDj8, and mDj11, fused to the Gal4 activation domain were cotransformed into yeast Y190 cells with expression vectors encoding K8 and K18 cDNAs fused to the Gal4 DNA-binding domain. The interactions were determined as described for B. D, identification of the coil II region of K18 that is sufficient for binding to Mrj. Y190 cells cotransformed with various pYTH9-K18 deletion mutants and pACT2-Mrj-F or pACT2-Mrj-C were selected in Trp/Leu-free medium and subjected to -galactosidase filter assay. Plus and minus signs are as described for B. Numbers refer to amino acid positions.

Direct Association of Mrj with K8/18 Filaments in Vitro-- Next we analyzed the in vitro direct association of Mrj with K8/18 filaments using a co-sedimentation assay (Fig. 2). As the separation of K18 and recombinant Mrj protein produced as a GST fusion protein (GST-Mrj) on Coomassie Brilliant Blue-stained gel after SDS-polyacrylamide gel electrophoresis was not clear (Fig. 2A), the samples were also detected with anti-GST antibody by immunoblotting to more clearly show the proportions of supernatant and precipitated GST-Mrj (Fig. 2B). Incubation in filament assembly buffer induced a rapid polymerization of K8 and K18 (Fig. 2A, lanes 1). Recombinant GST (which served as a control) did not sediment in the presence of K8/K18 (Fig. 2, A and B, lanes 3). In the absence of K8/18, GST-Mrj did not sediment (Fig. 2, A and B, lanes 4); but in the presence of K8/18, a substantial portion of GST-Mrj was precipitated with K8/18 (lanes 5). Because Mrj interacted with K18, but not with K8, in the two-hybrid analysis, the Mrj sedimenting with K8/18 was thought to be associated with the K8/18 polymer via direct binding to K18. In addition, Mrj did not sediment with vimentin (Fig. 2, A and B, lanes 6) or desmin (lanes 7), consistent with the results using the two-hybrid method.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Co-sedimentation of Mrj with K8/18. K8/18 filaments were assembled in the absence (lanes 1) or presence of GST (lanes 3) or GST-Mrj (lanes 5). As a control, only GST (lanes 2) or GST-Mrj protein (lanes 4) was incubated in the assembly buffer. Vimentin (lanes 6) or desmin (lanes 7) filaments were assembled in the presence of GST-Mrj. Samples of supernatant (S) and pellet (P) fractions were separated by SDS-polyacrylamide gel electrophoresis and then stained with Coomassie Brilliant Blue (A) or detected with anti-GST antibody by immunoblotting (B). Substantial portions of GST-Mrj sedimented with K8/18. GST-Mrj did not sediment with either vimentin or desmin under these conditions. The arrowheads and arrows indicate the positions of GST and GST-Mrj, respectively. Molecular size markers (in kilodaltons) are shown on the left.

Interaction of Mrj with K18 and Hsp/c70 in Vivo-- To further explore the interaction between Mrj and K18, polyclonal antibodies raised against Mrj were generated using the purified His₆-tagged Mrj protein as the immunogen. Affinity-purified rabbit anti-Mrj antibody specifically recognized a polypeptide with a relative molecular mass of 27 kDa in HeLa and T24 lysates (Fig. 3A). Preincubation of the antibody with recombinant His₆-tagged Mrj selectively inhibited the immunoreactivity (Fig. 3A). Using this antibody, we precipitated complexes containing Mrj proteins from 1% Triton X-100-soluble fractions of HeLa cell lysates (Fig. 3B). Mrj was immunoprecipitated with anti-Mrj antibody, but not by control rabbit IgG. K18 was co-immunoprecipitated with Mrj using anti-Mrj antibody. Because DnaJ/Hsp40 family proteins are known to interact with Hsp/c70, we examined whether Mrj was associated with Hsp/c70. Hsp/c70 was indeed detected in the Mrj immune complex. These results indicate that Mrj is associated with K18 and Hsp/c70 in vivo.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   In vivo association of Mrj with K18 and Hsp/c70. A, detection of Mrj in HeLa and T24 cells. Lysates from HeLa cells (lanes 1, 3, and 5) and T24 cells (lanes 2, 4, and 6) were stained with Coomassie Brilliant Blue (CBB) (lanes 1 and 2), detected with anti-Mrj antibody by immunoblotting (lanes 3 and 4), or immunostained with anti-Mrj antibody pre-absorbed by His-Mrj protein (lanes 5 and 6). Anti-Mrj antibody specifically recognized bands of ~27 kDa in samples obtained from HeLa and T24 cells. Molecular size markers (in kilodaltons) are shown on the left. B, interactions of Mrj with K18 and Hsp/c70 in vivo. Immunoprecipitates were prepared from lysates of HeLa cells using control rabbit IgG (lane 1) and anti-Mrj antibody (lane 2) as described under "Experimental Procedures." Each precipitate, separated into three parts, was subjected to immunoblot analysis with anti-Mrj, anti-K18, or anti-Hsp/c70 antibody.

Mrj Colocalizes with K8/18 Filaments in HeLa Cells-- To determine the intracellular distribution of Mrj and to determine if Mrj colocalizes with K8/18 filament networks, HeLa cells were fixed with 50% methanol and 50% acetone and double-stained with anti-Mrj polyclonal antibody and anti-K18 monoclonal antibody. Mrj was present in both the cytoplasm and nucleus (Fig. 4a). In the cytoplasm, Mrj was dispersed mainly in the perinuclear area and showed some filamentous patterns. There was a significant overlap between filamentous Mrj and K18 (Fig. 4, a-d), where Mrj was more remarkably associated with K8/18 filament bundles around perinuclear areas than with finer filament structures in the cell periphery. In addition, colocalization of Mrj with K8/18 filament networks appeared to be heterogeneous, and the extent of the overlap differed from cell to cell, suggesting the dynamic feature of the interaction between Mrj and K8/18 filaments. These associations of Mrj with K8/18 were also observed in other epithelial cell lines such as T24, Madin-Darby canine kidney, and COS-7 cells (data not shown). When we investigated whether Mrj colocalized with other major cytoskeletons, such as actin filaments and microtubules, double staining showed no apparent co-distribution of Mrj with actin filaments (Fig. 4, e-h) or with microtubules (i-l). In addition, Mrj did not colocalize with vimentin filaments in HeLa cells (data not shown).

View larger version (75K): [in this window] [in a new window]   Fig. 4.   Colocalization of Mrj and K8/18 filaments in HeLa cells. HeLa cells were double-stained with anti-Mrj antibody (a) and anti-K18 antibody (b). A merged image of a and b (c) and an enlargement of the area indicated by the arrow in c (d) are shown. The double immunofluorescence analysis of Mrj immunoreactivity (e) and actin immunoreactivity (f), an overlaid image of e and f (g), and magnification of the area indicated by the arrow in g (h) are shown. HeLa cells were also co-stained with anti-Mrj antibody (i) and anti-tubulin antibody (j). The superimposed image of i and j (k) and an enlargement of the area indicated by the arrow in k (l) are shown. Bars = 20 µm.

Effects of the Overexpression of Deletion Mutants of Mrj on K8/18 Filaments-- To further examine the function of Mrj, we determined which region of Mrj interacts with Hsp/c70 and K18 using the two-hybrid system. As shown in Fig. 5 (A and B), Mrj-F interacted with both K18 and Hsc70, but not with K8. Mrj-C (aa 99-242) strongly interacted with K18, but Mrj-N1 (aa 1-100) and Mrj-N2 (aa 1-146) did not bind to K18. In contrast, Mrj-N1 and Mrj-N2 bound to Hsc70, but Mrj-C did not interact with Hsc70. The interaction of Mrj-N2 with Hsc70 was stronger than that of Mrj-N1 with Hsc70, suggesting that in addition to the J domain, the glycine/phenylalanine-rich domain may be important for the tight association of Mrj with Hsp/c70. These results are consistent with a recent report of the characterization of the interaction between E. coli DnaJ and DnaK demonstrating, using surface plasmon resonance detection (BIAcore), that aa 1-76 of DnaJ bound to DnaK with somewhat lower affinity than did aa 1-108 of DnaJ (41). Taken together, the two-hybrid assay for the interaction between Mrj and K18 or Hsc70 shows that Mrj interacts with K18 through its C terminus and binds to Hsp/c70 via its N terminus-containing J domain. Based on these characterizations of the domains of Mrj, we speculated that the overexpression of Mrj-N2 might dominant-negatively inhibit the interactions between endogenous Mrj and Hsp/c70 and that the overexpression of Mrj-C might dominant-negatively inhibit the interaction between endogenous Mrj and K18. To check this idea, we transiently expressed Myc-tagged full-length Mrj (Myc-Mrj-F), Myc-Mrj-N2, and Myc-Mrj-C in HeLa cells and observed the effects of the expressed Mrj proteins on K8/18 filaments as well as actin filaments and microtubules. The expressed Myc-Mrj-F protein showed almost the same distribution as did the native Mrj protein (Fig. 5, C-E). The expressed Myc-Mrj-N2 protein was present predominantly in the nucleus as well as diffusely in the cytoplasm (Fig. 5, C-E). The expression of Myc-Mrj-N2 resulted in a pronounced disruption in the K8/18 filament network, although the expressed Myc-Mrj-N2 protein did not apparently colocalize with the collapsed K8/18 filaments around the nucleus (Fig. 5C, panels d-f). The expressed Myc-Mrj-C protein was present only in the cytoplasm and aggregated with K18, which resulted in the disruption of K8/18 filaments (Fig. 5C, panels g-i). The expression of Myc-Mrj-F or Myc-Mrj-C had no apparent effects on actin filaments (Fig. 5D), microtubules (Fig. 5E), and vimentin filaments (data not shown). In contrast, the expression of Myc-Mrj-N2 resulted in the disruption not only of K8/18 filaments (Fig. 5C, panels d-f), but also of actin filaments (Fig. 5D, panels d-f), microtubules (Fig. 5E, panels d-f), and vimentin filaments (data not shown). Almost the same phenotype was observed with other cell lines, including T24 and COS-7 cells (data not shown). Although the overexpression of Mrj-N2 would be initially expected to specifically inhibit the Mrj-Hsp/c70 interaction and to have effects on only K8/18 filaments, these results suggest that the overexpression of Myc-Mrj-N2 may inhibit other functions of Hsp/c70 chaperones. Consistent with this observation, Michels et al. (42) recently reported that coexpression of a truncated protein restricted to the J domain of Hsp40 had a dominant-negative effect on Hsp70-facilitated luciferase reactivation, suggesting that the overexpression of the J domain of Hsp40 might be used as an excellent tool to knock out Hsp70 function and might result in serious pathophysiological situations. Therefore, the phenotype induced by the overexpression of Myc-Mrj-N2 may be due to the inhibition of some or overall functions of Hsp/c70. In addition, as for the results of the overexpression of Myc-Mrj-C, we should note a possibility that the disruption of K8/18 filaments by the overexpression of Myc-Mrj-C is an artifact of peptide binding to the assembly domain of K18 rather than of the inhibition of the function of endogenous Mrj.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Analysis of Mrj deletion mutants. A, schematic representation of full-length Mrj and its deletion mutants. Numbers refer to amino acid positions. B, mapping of Mrj-K18 and Mrj-Hsc70 interaction domains using the yeast two-hybrid system. Expression vector pGBT9-Mrj-F, -Mrj-N1, -Mrj-N2, or -Mrj-C was cotransformed into yeast Y190 cells with pACT2-K8, -K18, or -Hsc70. The interactions were determined by -galactosidase assay as described in the legend to Fig. 1B. C-E, effects of expression of Myc-Mrj-F, Myc-Mrj-N2, or Myc-Mrj-C on K8/18 filaments (C), actin filaments (D), and microtubules (E) in HeLa cells. pRK5-Myc-Mrj-F (C-E, panels a-c), pRK5-Myc-Mrj-N2 (C-E, panels d-f), and pRK5-Myc-Mrj-C (C-E, panels g-i) vectors were transfected into HeLa cells. The transfected cells were fixed 16 h later and double-stained with anti-Myc antibody (C-E, panels a, d, and g) and anti-K18 antibody (C, panels b, e, and h), anti-actin antibody (D, panels b, e, and h), or anti-tubulin antibody (E, panels b, e, and h). The merged images of panels a and b (panels c), panels d and e (panels f), and panels g and h (panels i) are shown. Bars = 20 µm. G/F, glycine/phenylalanine-rich domain.

K8/18 Filament Organization Is Affected by Microinjection of Anti-Mrj Antibody-- The overexpression of Mrj deletion mutants indeed resulted in the disorganization of K8/18 filaments, but these results may not provide us with a conclusive answer for a role of Mrj as described above. To overcome this problem and to address an actual role of Mrj in K8/18 filament organization, microinjection of anti-Mrj antibody was carried out. Either anti-Mrj antibody or normal rabbit IgG was injected into HeLa cells; and 1 h later, cells were fixed and stained for K18, actin, or tubulin. Microinjection of normal rabbit IgG had no apparent effects on K8/18 filaments, actin filaments, and microtubules (data not shown). Microinjection of anti-Mrj antibody resulted in the disruption of K8/18 filaments (Fig. 6, a-c). In contrast, microinjection of anti-Mrj antibody did not have any effects on actin filaments (Fig. 6, d-f) or microtubules (g-i). These results clearly demonstrate that Mrj is involved in the organization of K8/18 filaments in vivo.

View larger version (97K): [in this window] [in a new window]   Fig. 6.   Microinjection of anti-Mrj antibody. One hour after microinjection of anti-Mrj antibody into HeLa cells, cells were fixed and then subjected to immunofluorescence study. The fixed cells were first incubated with Alexa 488-labeled anti-rabbit antibody to detect the injected anti-Mrj antibody and cells (a, d, and g). Next, the cells were double-stained for K18 (b), actin (e), or tubulin (h). Merged images of a and b (c), d and e (f), and g and h (i) are shown. Bars = 20 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we identified Mrj, a recently cloned DnaJ/Hsp40 family protein, as a K18-binding protein using the yeast two-hybrid system, biochemical analysis, and cell biological techniques. The human Mrj gene has been mapped to the chromosome 11q25 region, and the open reading frame of the gene encodes a protein of 242 amino acid residues containing a canonical J domain, a highly conserved 70-amino acid region at the N-terminal end of DnaJ/Hsp40 family proteins, and a glycine/phenylalanine-rich domain, which is also present in DnaJ/Hsp40 family proteins (Fig. 1A) (40). Mouse homozygous Mrj mutants died at mid-gestation due to a failure of chorioallantoic fusion at embryonic day 8.5, of which the molecular mechanism is unknown (39). We addressed that one of the specific functions of Mrj may be to regulate K8/18 filament organization.

The DnaJ/Hsp40 family proteins work together with the DnaK/Hsp70 class of chaperones, which comprise a set of abundant cellular machines that assist a large variety of protein folding processes in almost all cellular compartments (43, 44). The J domain of DnaJ/Hsp40 family proteins is essential to stimulate the weak intrinsic ATPase activity of Hsp70 proteins (43, 44). Since Hsp70 proteins have a very broad substrate specificity (45-47), it has been proposed that some of the DnaJ/Hsp40 family proteins act as specialized co-chaperones that recruit an Hsp70 protein to a specific set of substrates (48-50). Cytosolic Hsp/c70 has been reported to be associated with K8/18 via direct binding to K8, although the nature of the relationship between Hsp/c70 and K8/18 remains uncertain (30). We showed here that Mrj was associated in vitro with K8/18 filaments via direct binding to K18 using a co-sedimentation assay. In a two-hybrid assay, Mrj interacted strongly with K18, but not with other IF proteins, including K5, K8, K14, vimentin, GFAP, and desmin. Among the interactions between DnaJ/Hsp40 family proteins and various IF proteins that we have thus far tested using the two-hybrid method, only the Mrj-K18 interaction was apparently positive. Mrj was associated in vivo with K18 and Hsp/c70 and colocalized with K8/18 filaments. Mrj interacted with Hsc70 via its N terminus-containing J domain and bound to K18 through its C terminus. These results suggest that Mrj may act as a K18 (K8/18)-specific adaptor protein to link K18 (K8/18) to the Hsp/c70 chaperone. Furthermore, microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, indicating that Mrj plays an indispensable role in the organization of K8/18 filaments in vivo.

In HeLa cells, the overexpression of the N-terminal region of Mrj (Myc-Mrj-N2) resulted in the disruption not only of K8/18 filaments, but also of actin filaments, microtubules, and vimentin filaments. Because Mrj-N2 can interact with Hsc70, but not with K18, the overexpressed Myc-Mrj-N2 protein would be initially expected to dominant-negatively inhibit the specific interaction between endogenous Mrj and Hsp/c70. As described above, Michels et al. (42) recently reported that the overexpression of a truncated protein restricted to the J domain of Hsp40 showed dominant-negative effects on the chaperone activity of Hsp70 in a living cell. Therefore, the effects of the overexpression of Myc-Mrj-N2 on major cytoskeletal systems observed in this study might be caused by the disturbance of some or overall functions of the Hsp/c70 chaperone system rather than by the specific inhibition of the Mrj-Hsp/c70 interaction. If this is the case, these results indicate a possibility that Hsp/c70 chaperones may play an important role in the regulation of the organization of actin filaments, microtubules, and vimentin filaments as well as K8/18 filaments. In addition, it is tempting to speculate that there may exist some specific DnaJ/Hsp40 family proteins that link actin, tubulin, or various IF proteins to Hsp/c70 (Fig. 7). In support of this idea, there is a growing body of evidence suggesting an intimate relationship between the cytoskeleton and molecular chaperones, including the Hsp/c70 family (51). In S. cerevisiae, it was genetically demonstrated that Ssa1p (yeast cytosolic Hsp70) and Ydj1p (yeast cytosolic DnaJ homologue) play a role in the regulation of microtubule formation (52). It was also reported that in human gastric cancer cells, overexpressed BAG-1, a negative regulator of Hsp/c70 chaperone activity, colocalizes with keratin filaments as well as actin filaments and promoted cell migration (53). Taken together, these observations will shed new light on a possible role of the Hsp/c70 chaperone in the regulation of cytoskeletons.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Working hypothesis of the regulation of major cytoskeletons by the Hsp/c70 chaperone and DnaJ/Hsp40 family co-chaperones.

B-crystallin and Hsp27, members of the small Hsp family, were reported to be associated with both soluble and filamentous IF proteins, including GFAP, vimentin, and keratin (54, 55). The in vitro polymerization of GFAP and vimentin is repressed by B-crystallin (54) as well as by Hsp27 (55). A physiological role for B-crystallin with IFs was further demonstrated by a recent report describing that a mutation in the B-crystallin gene causes desmin aggregation in certain myopathies called desmin-related myopathies (56). As for the pathological aggregation of K8/18, it is well known that Mallory bodies in the livers of human patients with alcoholic hepatitis and of griseofulvin-treated mice are the cytoplasmic accumulation of K8/18-containing aggregates (57-60). As described earlier, old K18 null mice developed a distinctive liver pathology with abnormal hepatocytes containing K8-positive aggregates that resembled Mallory bodies (24). The finding that K8/18 filament organization may be regulated by the Mrj-Hsp/c70 chaperone will provide a new clue to dissect the mechanism of the formation of Mallory bodies.

Assembly and disassembly of IF organization are regulated by the site-specific phosphorylation state of IFs, and these phosphorylations are spatially and temporally regulated during cellular events, including mitosis (9, 10, 11, 61). When phosphorylated by purified protein kinase C, calmodulin-dependent protein kinase, or cAMP-dependent kinase, K8/18 filaments reconstituted in vitro undergo complete disassembly, and there is a significant release of soluble K8 and K18 proteins from the keratin filaments (12). It has been reported that the increase in K8/18 phosphorylation is associated with the binding of the soluble fraction of K8/18 to 14-3-3 proteins (33). Because Mrj is associated with both soluble and filamentous K8/18, it is likely that the phosphorylation state of K8/18 may not directly affect the association of Mrj with K8/18. The relationship between the phosphorylation-dependent and Mrj-dependent regulation of K8/18 filaments needs to be investigated in the future.

In conclusion, we are the first to report that human Mrj proteins directly interact with K18 and might regulate K8/18 filament organization as K18-specific co-chaperones working together with Hsp/c70. These observations will pave the way toward further research on K8/18 filament organization as well as understanding the pathological mechanisms of Mallory body formation.

	ACKNOWLEDGEMENTS

We thank H. Eto for kindly providing the pGEX-2TH-K8 and -K18 plasmids, E. Fuchs for kindly providing the pET8c-K5 and -K14 plasmids, N. Imamoto for providing a plasmid harboring human Hsc70 cDNA, T. Saito and A. Hattori (National Institute of Radiological Sciences, Chiba, Japan) for helpful suggestions regarding Mrj, S. Ando and R. Gohara (Saga Medical School, Saga, Japan) for helpful discussions, Toshimichi Yoshida (Mie University School of Medicine) for suggestions, and K. Nagata (our laboratory) for technical advice and helpful discussions. We are grateful to M. Ohara for a critique of the manuscript.

	FOOTNOTES

^* This work was supported in part by grants-in-aid for scientific research and cancer research from the Ministry of Education, Science, Sports, and Culture of Japan; by the Japan Society for Promotion of Science Research for the Future; by a grant-in-aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan; by a grant from Bristol-Myers-Squibb; and by the Princess Takamatsu Cancer Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Div. of Biochemistry, Aichi Cancer Center Research Inst., 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681, Japan. Tel.: 81-52-762-6111 (ext. 7020); Fax: 81-52-763-5233; E-mail: minagaki@aichi-cc.pref.aichi.jp.

Published, JBC Papers in Press, August 22, 2000, DOI 10.1074/jbc.M003492200

	ABBREVIATIONS

The abbreviations used are: IFs, intermediate filaments; K, keratin; aa, amino acids; GST, glutathione S-transferase; GFAP, glial fibrillary acidic protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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