Báo cáo khoa học: A client-binding site of Cdc37 ppt

A client-binding site of Cdc37 Kazuya Terasawa and Yasufumi Minami Department of Biophysics and Biochemistry, and Undergraduate Program for Bioinformatics and Systems Biology, Graduate School of Science, University of Tokyo, Japan Molecular chaperones are required for the correct fold- ing of many proteins inside cells, despite their confor- mations being predetermined by their own amino acid sequences, because de novo protein synthesis proceeds in a directional manner from the N-terminus and encounters a cellular milieu that is crowded with macromolecules [1]. Although Hsp90 is abundant and highly conserved among species, its structure and func- tional mechanism have been unveiled only quite recently [2–7]. Whereas Hsp70 and chaperonin act as general chaperones in the early stage folding of newly synthesized proteins [1,8,9], Hsp90 takes part in the folding of client proteins at a later stage of maturation [2–7]. In addition, Hsp90 client proteins seem to be restricted to cell-signaling molecules, such as steroid hormone receptors and protein kinases [2–7]. It is now appreciated that Hsp90 performs the chaperone function in a manner dependent on its own ATPase activity, serving as an ATPase-driven molecular clamp that binds and releases client proteins in a closed and open state, respectively, this conformational trans- ition being controlled by ATP binding and hydrolysis [2–7]. Moreover, this ATPase-dependent chaperone cycle is cooperatively tuned by various co-chaperones [2–7]. Cdc37 ⁄ p50 is one Hsp90 co-chaperone and is characterized as a protein kinase-specific cofactor for Hsp90 [10–12], because Cdc37 interacts both physically and genetically with a variety of protein kinases, inclu- ding pp60 v-src [13], Raf-1 [14] and Cdk4 [15,16]. Cdc37 binds directly to Hsp90 [17–19]; a recent crystallogra- phic study found that the C-terminal domain of Cdc37 interacts with the N-terminal ATP-binding domain of Hsp90 [20]. In the crystal structure, Cdc37 binds to the open face of the Hsp90 N-terminal domain, interfering with conformational changes of Hsp90 crucial for its ATPase activity; this accords well with the finding that Cdc37 inhibits Hsp90 ATPase activity [21]. Concomit- ant with the binding to Hsp90, Cdc37 can associate Keywords Cdc37, Hsp90, protein kinase, Raf-1 Correspondence Y. Minami, Department of Biophysics and Biochemistry, and Undergraduate Program for Bioinformatics and Systems Biology, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan Fax: +81 3 5841 3047 Tel: +81 3 5841 3047 E-mail: yminami@biochem.s.u-tokyo.ac.jp (Received 28 June 2005, revised 23 July 2005, accepted 26 July 2005) doi:10.1111/j.1742-4658.2005.04884.x The molecular chaperone Hsp90 is distinct from Hsp70 and chaperonin in that client proteins are apparently restricted to a subset of proteins categor- ized as cellular signaling molecules. Among these, many specific protein kinases require the assistance of Hsp90 and its co-chaperone Cdc37 ⁄ p50 for their biogenesis. A series of Cdc37 deletion mutants revealed that all mutants capable of binding Raf-1 possess amino acid residues between 181 and 200. The 20-residue region is sufficient and, in particular, a five-residue segment (residue 191–195) is essential for binding to Raf-1. These five resi- dues are present in one a helix (residues 184–199) in the middle of Cdc37, which is unexpectedly nested within the Hsp90-interacting domain of Cdc37, which was recently determined by crystallography, but does not seem to contribute to direct contact with Hsp90. Furthermore, an N-ter- minally truncated mutant of Cdc37 composed of residues 181–378 was shown to bind the N-terminal portion of Raf-1 (subdomains I–IV). This mutant can bind not only other Hsp90 client protein kinases, Akt1, Aurora B and Cdk4, but also Cdc2 and Cdk2, which to date have not been shown to physically interact with Cdc37. These results suggest that a region of Cdc37 other than the client-binding site may be responsible for discriminating client protein kinases from others. Abbreviation GST, glutathione S-transferase; IP, immunoprecipitation; Knd, kinase domain; WB, western blot. 4684 FEBS Journal 272 (2005) 4684–4690 ª 2005 FEBS with protein kinases [14–19,22–24], in particular, with their N-terminal lobes [25–28]. Thus, one role of Cdc37 is thought to be client recruitment to Hsp90; however, this view is simplistic [11,12]. Cdc37 has the potential to exhibit chaperone activity independent of Hsp90 [22,23,29–31] and its repertoire of client proteins stret- ches beyond the protein kinases [26,32,33]. Even though our knowledge of Hsp90 has increased dramatically and is currently being updated further [2–7], the whole spectrum of Hsp90 client proteins and the comprehensive mechanism of the Hsp90 chaperone cycle remain obscure. To challenge these questions, we analyzed a set of Cdc37 deletion mutants and eventu- ally identified a 20-residue region of Cdc37 (residues 181–200) as a client-binding site, in which five residues (residues 191–195) are important for client binding and are located on an a helix in the middle of Cdc37. The helix is embedded in the Hsp90-binding domain of Cdc37 in the primary structure; however, it is not involved in interactions with Hsp90 [20]. We found that an N-terminally truncated mutant of Cdc37 con- taining residues 181–378, but not the full-length Cdc37, is able to associate with Cdc2 and Cdk2 (which have not been reported to physically interact with Cdc37) in addition to the well-known Hsp90 client protein kinases, Raf-1, Akt1, Aurora B and Cdk4. These findings may suggest how Cdc37 ⁄ Hsp90 distin- guishes a limited set of protein kinases from others. Results and Discussion Cdc37 deletion mutants We analyzed a series of Cdc37 deletion mutants expressed in COS7 cells (Fig. 1A) to identify the client- binding site. Both the C- and N-terminally truncated FLAG-tagged Cdc37 (hereafter called FLAG–Cdc37) mutants, FLAG–Cdc37(1–200), and FLAG–Cdc37(181– 378), respectively, bind the protein kinase domain of Raf-1, as shown in Fig. 1B [immunoprecipita- tion (IP): a-FLAG, middle panel]. Consequently, an overlapping region (residues between 181 and 200) was suggested to be the client-binding site of the Raf-1 kinase domain; this was reinforced by the fact that FLAG–Cdc37(1–180) and FLAG–Cdc37(201–378), nei- ther of which contain the above-mentioned region, were unable to bind the kinase domain (Fig. 1B). Further- more, these observations were corroborated by an inverse immunoprecipitation experiment using the Raf-1 kinase domain (Fig. 1B, IP: a-Myc, right). However, C-terminally truncated Cdc37 (residues 1–163) has previously been reported to bind Raf-1 [18]. We per- formed a similar experiment using the N- and C-terminal portions of Cdc37, namely FLAG–Cdc37(1– 163) and FLAG–Cdc37(164–378), respectively, and found that FLAG–Cdc37(164–378) could bind Raf-1 to a similar extent as the full-length Cdc37, whereas B FL 1-276 1-200 1-180 181-378 201-378 IP: α-Myc whole FLAG-Cdc37 FL 1-276 1-200 1-180 181-378 201-378 α-FLAG WB: α-Myc * * IP: α-FLAG FL 1-276 1-200 1-180 181-378 201-378 A 1 378 FL 1-276 1-200 1-180 181-378 Knd + + + - + -201-378 Fig. 1. The residues between 181 and 200 of Cdc37 are required for binding the Raf-1 kinase domain. (A) Primary structures of the full-length Cdc37 (FL) and its deletion mutants with numbers corresponding to the first and last residue, and their binding activities toward the Raf-1 kin- ase domain (Knd) are schematically illustrated. The residues between 181 and 200 are shaded. (B) The Myc-tagged kinase domain of Raf-1 and the full-length Cdc37 or each Cdc37 deletion mutant were coexpressed in COS7 cells (whole) and the obtained cell extracts were subjec- ted to immunoprecipitation with anti-FLAG (IP: a-FLAG) or anti-Myc (IP: a-Myc) monoclonal antibody, followed by immunoblotting with both anti-Myc and anti-FLAG polyclonal antibodies (WB: a-Myc and a-FLAG). Asterisks indicate nonspecific bands appearing in every lane. K. Terasawa and Y. Minami A client-binding site of Cdc37 FEBS Journal 272 (2005) 4684–4690 ª 2005 FEBS 4685 FLAG–Cdc37(1–163) was hardly coimmunoprecipita- ted with Raf-1 (data not shown); these results are consis- tent with those shown in Fig. 1, however, they do not agree with previously reported results [18]; we are not able to interpret this difference at present. Next, we selected one deletion mutant FLAG– Cdc37(181–378) to further delineate the tentative client- binding region of Cdc37. Three protein kinases (Fig. 2A), Akt1 [34], Aurora B [35] and Cdk4 [15,16], which have previously been reported to bind to Cdc37, were all bound to FLAG–Cdc37(181–378) (Fig. 2B). We repeated the experiment with the kinase domains of Akt1 and Aurora B instead of the whole molecules, omitting Cdk4 because it is composed almost solely of a kinase domain (Fig. 2A). It was clearly shown that the kinase domains of both Akt1 and Aurora B bound to FLAG–Cdc37(181–378) (Fig. 2C). Moreover, endogenous Raf-1 (not ectopically expressed Raf-1) in COS7 cells interacted with this deletion mutant as strongly as the full-length Cdc37 (see below). When the kinase domain of Raf-1 was divided between subdomains IV and V [36] into the N- and C-terminal portions and each was fused to Myc–gluta- thione S-transferase (GST), as shown in Fig. 3A, the N-terminal portion of Raf-1 (subdomains I–IV), but not the C-terminal portion (subdomains V–XI), was bound to FLAG–Cdc37(181–378) (Fig. 3B). Our results are consistent with previous studies; Cdc37 interacts with protein kinases via their N-terminal lobes [25–28]. It was shown that the deletion mutant of Cdc37, FLAG–Cdc37(181–378), is able to bind the client protein kinases; therefore, it contains a client-binding site. B C B a ror u A 4kdC 1tkA - IP: α-Myc whole Aurora B Cdk4 Akt1 181-378 * WB: α-FLAG α-Myc Myc-Kinase IP: α-Mycwhole WB: α-FLAG α-Myc * B aro r uA 1 tkA - B a r or uA 1t k A -Myc-Knd Akt1 Cdk4 Aurora B 3041 5 295 345 1 76 327 4801 149 409 Knd A B a r oru A 4k d C 1 tkA - Fig. 2. FLAG–Cdc37(181– 378) binds three known Cdc37 client pro- tein kinases, Akt1, Aurora B and Cdk4. (A) Primary structures of Akt1, Aurora B and Cdk4 are schematically drawn with residue numbers, where in particular, their kinase domains (Knd, light lines) are discriminated from other regions (dark lines). (B) FLAG– Cdc37(181–378) was expressed alone (–) or coexpressed with Myc-tagged kinases (Myc-Kinase) as indicated in COS7 cells and the cell lysates (whole) were immunoprecipitated with anti-Myc monoclonal antibody (IP: a-Myc), followed by immunoblotting with the indicated polyclonal antibodies. Asterisks indicate nonspecific bands appearing in every lane. (C) Myc-tagged kinase domains (Myc-Knd) were used instead of their whole molecules, and the obtained immunoprepitates were analyzed by immunostaining as described in (B). α-Myc Knd I-IV Myc-GST α-FLAG whole empty GST pull-down GST pull-down V-XI WB: B I-IV V-XI Myc-GST fusion fragment Myc GST A Raf-1 Knd 614349 414/415 Fig. 3. Cdc37 binds the N-teminal portion of Raf-1. (A) (Upper) Pri- mary structure of the kinase domain of Raf-1 (Knd), and its N- and C-terminal portions (I–IV and V–XI, respectively) are schematically depicted with residue numbers. (Lower) Schematic drawing of the Myc-GST fusion construct is shown; either Knd, the N- or C-ter- minal portion of Raf-1 was inserted at a position indicated by ‘frag- ment’. (B) FLAG–Cdc37(181– 378) and, Myc–GST alone (empty) or fused with Knd, I–IV or V–XI were coexpressed in COS7 cells. The cell lysates (whole) were pulled down with glutathione beads (GST pull-down), after which immunoblotting with anti-FLAG or anti-Myc polyclonal antibody was performed (WB: a-FLAG and a-Myc). A client-binding site of Cdc37 K. Terasawa and Y. Minami 4686 FEBS Journal 272 (2005) 4684–4690 ª 2005 FEBS The 20-residue region of Cdc37 is a client-binding site Because the above results infer that the 20-residue region of Cdc37 is essential for the binding of a client protein kinase, we tested whether the peptide (residues 181–200 of Cdc37) conjugated to FLAG–GST (Fig. 4A) was able to bind the kinase domain of Raf-1. As shown in Fig. 4B, immunoprecipitaton with both anti-FLAG (IP: a-FLAG; for FLAG–GST–peptide) and anti-Myc (IP: a-Myc; for a Myc-tagged kinase domain of Raf-1) monoclonal antibodies proved that this peptide is capable of binding the Raf-1 kinase domain, which was further confirmed for the kinase domains of Akt1 and Aurora B, and full-length Cdk4 (Fig. 4C). Thus, it could be concluded that the 20-resi- due region of Cdc37 is sufficient for the binding of client protein kinases. To specify the required residues in the peptide, alanine-scanning and deletion mutagenesis of the two-residue region were performed (Fig. 5A). Alanine- scanning mutagenesis abolished the ability of mutant 3A to bind the Raf-1 kinase domain, and the ability of mutant 4A to bind the Raf-1 kinase domain was remarkably decreased (Fig. 5B). Deletion mutant N10 lost its binding activity, but two mutants, M10 and C10, retained it (Fig. 5C). Taken together, these results support the conclusion that a five-residue segment, VIWCI (residues 191–195), is maximally required for interaction with the kinase domain of Raf-1. This segment resides in an a helix composed of residues between 184 and 199, which was recently A 181 200 ELVC ETANYLV IWC I DLEVE FLAG GST peptide B α-FLAG WB: α-Myc whole IP: α-Myc IP: α-FLAG * Aurora B-Knd Cdk4 (FL) WB: α-FLAG α-Myc whole IP: α-Myc Akt1-Knd C - * Aurora B-Knd Cdk4 (FL) Akt1-Knd - Myc-Knd Fig. 4. Cdc37 peptide (residues between 181 and 200) fused with FLAG–GST binds kinase domains. (A) A primary structure of the FLAG–GST–peptide fusion is schematically illustrated, with the pep- tide sequence from 181 to 200 of Cdc37. (B) The Myc-tagged kin- ase domain of Raf-1 and FLAG–GST fused with nothing (i.e. empty) (e) or the peptide (p) were coexpressed in COS7 cells. Cell extracts were prepared (whole) and subjected to immunoprecipitation with anti-FLAG and anti-Myc monoclonal antibodies (IP: a-FLAG and a-Myc), followed by immunoblotting with anti-FLAG and anti-Myc polyclonal antibodies (WB: a-FLAG and a-Myc). Asterisks indicate nonspecific bands appearing in every lane. (C) FLAG–GST–peptide fusion protein was expressed alone (–) or coexpressed with Myc-tagged kinase domains of Akt1 or Aurora B, or Myc-tagged full-length Cdk4 (FL) in COS7 cells and the obtained immunoprecipi- tates were analyzed by immunostaining as described in (B). α-FLAG WB: α-Myc whole FLAG-GST IP: α-FLAG B 1A 2A 3A 4A 5Awt 1A 2A 3A 4A 5Awt whole IP: α-FLAG N10 M10 C10wt C α-FLAG WB: α-Myc FLAG-GST wt N10 M10 C10 A 181 200 wt 1A 2A 3A 4A 5A N10 M10 C10 LVCEETANYLVIWCIDLEVE AAAAETANYLVIWCIDLEVE LVCEAAAAYLVIWCIDLEVE LVCEETANAAAAWCIDLEVE LVCEETANYLVIAAAALEVE LVCEETANYLVIWCIDAAAA LVCEETANYL TANYLVIWCI VIWCIDLEVE Fig. 5. Five residues of Cdc37 are essential for the binding of the Raf-1 kinase domain. (A) Peptide sequences fused to FLAG–GST are shown; wt: a wild-type peptide; 1A)5A: five different alanine- scanning mutant peptides (four consecutive residues changed to alanine are underlined); N10, M10 and C10: 10-residue truncation mutant peptides. The five most important residues, VIWCI, are sha- ded. (B, C) The Myc-tagged kinase domain of Raf-1 and each FLAG–GST–peptide indicated were coexpressed in COS7 cells. The obtained cell lysates (whole) were immunoprecipitated with anti- FLAG monoclonal antibody (IP: a-FLAG) and subsequently immuno- stained with anti-Myc (for Myc-Knd) and anti-FLAG (for FLAG–GST– peptide) polyclonal antibodies (WB: a-Myc and a-FLAG). K. Terasawa and Y. Minami A client-binding site of Cdc37 FEBS Journal 272 (2005) 4684–4690 ª 2005 FEBS 4687 determined using crystallography [20], and unexpect- edly, is nested within the Hsp90-binding region in the primary structure. However, the helix does not partici- pate in physical interaction with Hsp90 [20]. The N-terminally deleted mutant of Cdc37 binds Cdc2 and Cdk2 We wondered whether the N-terminally deleted mutant of Cdc37, FLAG–Cdc37(181–378), would bind protein kinases other than well-known client protein kinases such as Raf-1. Yeast Cdc28 (Cdc2 homolog) has been reported to interact genetically with Cdc37 [37,38] and their interaction was shown in a yeast two-hybrid sys- tem [25], however, Cdc2 did not appear to physically associate with Cdc37 [16]. As shown in Fig. 6 [western blot (WB): a-Cdc2], FLAG–Cdc37(181– 378) was able to bind endogenous Cdc2 in COS7 cells; however, full- length Cdc37 could not, which is compatible with the above study [16]. More surprisingly, FLAG– Cdc37(181–378) bound endogenous Cdk2 (Fig. 6, WB: a-Cdk2), whose interaction with Cdc37 was not detec- ted in a previous study [16]; indeed, Cdk2 was invisible in the immunoprecipitate of the full-length Cdc37 in this study also (Fig. 6). Thus, although Cdc37 selectively binds a subset of protein kinases, its N-terminally deleted mutant FLAG–Cdc37(181–378), which retains a binding site toward the known client protein kinases, becomes competent to bind protein kinases that do not appear to interact with full-length Cdc37. The data imply that many, if not all, protein kinases may possess similar sequences capable of interacting with the Cdc37 client- binding site determined in this study; this is conceiv- able because protein kinases are quite similar as far as the architecture of the catalytic domains is concerned [39]. Therefore, it must be clarified how Cdc37 prefer- ably distinguishes a limited set of protein kinases from others. The truncated region of FLAG–Cdc37(181– 378), i.e. the N-terminal portion of Cdc37 (residues 1–180), might be critically committed to its client selec- tion and binding, which is possibly in line with other studies [24,40–43]. Experimental procedures Cell culture and transfection COS7 cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium containing 10% (v ⁄ v) fetal bovine serum. Cells were transfected with Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Plasmid construction Full-length cDNAs of human Cdc37, Aurora B and Cdk4 were synthesized by PCR from mRNA isolated from HeLa cells. Plasmids used in this study (pcDNA3Myc1, pcDNA3- FLAG1, SRa and SRa-MycGST) were supplied by E. Nishida (Kyoto University, Japan). Full-length and var- ious mutant constructs of Cdc37, Aurora B and Cdk4 were produced by PCR with the addition of a BamHI site at the 5¢-end and an EcoRI site following a stop codon (TGA) at the 3¢-end, and each was ligated to either the pcDNA3- Myc1 or pcDNA3FLAG1 plasmid cut with both BamHI and EcoRI. The BamHI fragments of human Raf-1 cDNA (provided by E. Nishida) and human Akt1 cDNA (provi- ded by Y. Gotoh of The University of Tokyo, Japan) were inserted into the BamHI site of the pcDNA3Myc1 plasmid. Full-length, and the N- and C-terminally divided portions of Raf-1 (subdomains I–IV and V–XI) were produced by PCR with the addition of a BamHI site at the 5¢-end and an EcoRI site following a stop codon (TGA) at the 3¢-end, and each was ligated to the SRa–MycGST plasmid cut with both BglII and EcoRI. The oligonucleotide for a FLAG epitope tag was introduced into the SRa plasmid to obtain SRa–FLAG1. A coding region of GST was produced by PCR with the SRa–MycGST plasmid used as a template, concomitantly adding a BamHI and BglII site at the 5¢- and 3¢-end, respectively, and then was ligated to the BglII site of the SRa–FLAG1 plasmid, yielding SRa– FLAG–GST. To make constructs for GST–peptide fusion proteins, oligonucleotides corresponding to peptide sequences were inserted into the SRa–FLAG–GST plasmid. All constructs were confirmed by DNA sequencing. WB: α-Raf-1 α-Cdc2 α-Cdk2 whole IP: α-FLAG FL 181-378 α-FLAG FL FL 181- 378 181- 378 FLAG-Cdc37 Fig. 6. FLAG–Cdc37(181– 378) binds endogenous Raf-1, Cdc2 and Cdk2. FLAG-tagged full-length Cdc37 (FL) and FLAG–Cdc37(181– 378) were expressed in COS7 cells and the cell lysates (whole) were immunoprecipitated with anti-FLAG monoclonal antibody (IP: a-FLAG), followed by immunoblotting with the indicated polyclonal antibodies. A client-binding site of Cdc37 K. Terasawa and Y. Minami 4688 FEBS Journal 272 (2005) 4684–4690 ª 2005 FEBS Immmunoprecipitation and immunoblotting Cells were lyzed with lysis buffer containing 20 mm Hepes, pH 7.5, 1 mm MgCl 2 ,1mm EGTA, 150 mm NaCl, 1% (v ⁄ v) Nonidet P-40 and 1% (v ⁄ v) Proteinase Inhibitor Cocktail (Sigma, St. Louis, MO). To immunoprecipitate Myc-tagged proteins, cell lysates were mixed with c-Myc (9E10) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4 °C and further incubated in the pres- ence of protein G Sepharose (Amersham Biosciences, Piscataway, NJ) with gentle rotation for 2 h at 4 °C. FLAG-tagged proteins were immunoprecipitated by incuba- tion with anti-FLAG M2-Agarose (Sigma) for 2 h at 4 °C. GST-tagged proteins were pulled down by incubation with glutathione Sepharose 4B (Amersham Biosciences) for 2 h at 4 °C. The beads were collected by centrifugation and washed three times with lysis buffer. The obtained proteins were separated by SDS ⁄ PAGE and analyzed by immuno- blotting. 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A client-binding site of Cdc37 Kazuya Terasawa and Yasufumi Minami Department of Biophysics and Biochemistry, and Undergraduate Program for Bioinformatics