Human Muts Homologue MSH4 Physically Interacts with Von Hippel-Lindau Tumor Suppressor-Binding Protein 11

[CANCER RESEARCH 63, 865–872, February 15, 2003] Human MutS Homologue MSH4 Physically Interacts with von Hippel-Lindau Tumor Suppressor-binding Protein 11

Chengtao Her,2 Xiling Wu, Michael D. Griswold, and Feng Zhou School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-4660 [C. H., X. W., M. D. G.], and Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 [F. Z.]

ABSTRACT human MMR genes are linked to the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic tumors Increasing evidence indicated that the protein factors involved in DNA associated with microsatellite instability (1). Eukaryotic MutS mismatch repair (MMR) possess meiotic functions beyond the scope of DNA mismatch correction. The important roles of MMR components in homologous proteins MSH2, MSH3, and MSH6 are proteins meiotic processes have been highlighted by the recent identification of two known to participate in DNA MMR through the actions of their additional members of the mammalian MutS homologs, MSH4 and heterodimeric complexes consisting of either MSH2-MSH3 or MSH5. Mammalian MSH4 and MSH5 proteins form a heterodimeric MSH2-MSH6, in which the MSH2-MSH6 heterodimer recognizes complex and play an important role in the meiotic processes. As a step both single-base mismatches and small loops formed by insertions forward to the understanding of the molecular mechanisms underlying or deletions in the DNA, whereas the MSH2-MSH3 heterodimer the roles of these two mammalian MutS homologues, here we have iden- only recognizes small insertions and deletions (3, 4). tified von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 Recent evidence demonstrates that eukaryotes contained a sep- (VBP1) as an interacting protein partner for human MSH4 (hMSH4). In addition, we have characterized a hMSH4 splicing variant (hMSH4sv) arate and functionally related group of MutS homologues including encoding a truncated form of hMSH4. The protein encoded by hMSH4sv MSH4 and MSH5 (5–10). Diploid Saccharomyces cerevisiae lack- was unable to interact with hMSH5, but it retained the capacity to interact ing the MSH4 or MSH5 gene display decreased levels of spore with VBP1. It is conceivable that hMSH4 and hMSH4sv can carry out viability and reciprocal exchange between homologous chromo- different but overlapping functions by differential protein interactions, somes, along with an increase in nondisjunction of homologous and, therefore, hMSH4sv might represent a separation-of-function alter- chromosomes at meiosis I (5, 6). Similar to their yeast homo- native form of the hMSH4 protein. hMSH4 and VBP1 proteins were logues, mammalian homologous MSH4 and MSH5 proteins are colocalized in mammalian cells. Three-hybrid analysis suggested that VBP1 could compete with hMSH5 for the binding of hMSH4. Thus, also found to interact and form a heterodimeric structure (9–13). In hMSH4 may be involved in diverse cellular processes through interaction contrast to MSH5, the expression of human and mouse MSH4 is with different protein partners, and the levels of VBP1 protein expression relatively restricted to meiotic tissues (7, 10). Examination of in cells could potentially affect the availability of the hMSH4-hMSH5 mouse gene knockout models indicates that Msh4 and Msh5 act in hetero-complex. the same pathway during meiosis, and mice carrying the disrupted Msh4 or Msh5 gene display defective chromosome synapsis, resulting in testicular and ovarian degeneration and, therefore, male INTRODUCTION and female sterility (14–16). In contrast, yeast MSH4 and MSH5 DNA repair mechanisms are involved in both mitotic and mei- are not essential for the completion of meiotic processes (5, 6), otic cell divisions, and the integrity of genetic information passed raising the possibility that the mammalian homologous proteins from parental cells to daughter cells is controlled by multiple may have evolved to carry out additional cellular functions. It is cellular proteins involved in cell-cycle regulation, DNA replica- conceivable that mammalian MSH4-MSH5 heterodimer functions tion, DNA repair, and chromosome segregation. Among many in conjunction with the MLH1-PMS2 complex, as supported by the DNA repair pathways, DNA MMR3 defines one of the most fact that both MLH1 and PMS2 have meiotic functions, and by the important molecular mechanisms in maintaining the faithful trans- recent observation that hMSH4 physically interacts with human mission of genetic information during DNA replication. Mamma- MLH1 (17). However, the effect of Msh4 or Msh5 deficiency on lian MutS homologues represent important evolutionary conserved the complete disruption of ovarian development is unique among components involved in multiple biological functions such as DNA genes known to function in meiotic processes, raising the possi- MMR, mitotic and meiotic recombination, and cellular responses bility that mammalian MSH4 and MSH5 could be involved in to DNA damages (1). The MMR process is initiated with the cellular pathways other than its role in homologous recombination. recognition and binding of the mismatched nucleotides by het- However, the protein factors that may act together with hMSH4 erodimeric MutS homologous proteins (2). Mutations in several and hMSH5 in cellular pathways are unknown at present time. To gain a better understanding of the biological processes associ- Received 8/6/02; accepted 12/13/02. ated with hMSH4 in humans, we report here the characterization of a The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with hMSH4sv and the identification of VBP1 as an interacting protein for 18 U.S.C. Section 1734 solely to indicate this fact. both hMSH4 and hMSH4sv. VBP1 was first identified as a binding 1 Supported in part by an American Cancer Society Institutional Research grant. 2 To whom requests for reprints should be addressed, at School of Molecular Bio- factor for VHL tumor suppressor protein in humans (18). Homologues sciences and Center for Reproductive Biology, P. O. Box 644660, Washington State of VBP1 in other mammals and yeast were highly conserved; VBP1 University, Pullman, WA 99164-4660. Phone: (509) 335-7537; Fax: (509) 335-9688; proteins in human and mouse are 100% identical in amino acid E-mail: [email protected]. 3 The abbreviations used are: MMR, mismatch repair; hMSH4, human MSH4; hMSH4sv, sequences (18–20). Although the precise functions of VBP1 are hMSH4 splicing variant; VHL, von Hippel-Lindau; VBP1, VHL tumor suppressor-binding elusive at present time, it has been shown that VBP1 plays an essential protein 1; IPTG, isopropyl-1-thio-␤-D-galactopyranoside; BD, binding domain; AD, activating domain; ORF, open reading frame; UTR, untranslated region; GST, glutathione S-transferase; role in promoting the formation and assembly of functional tubulins, SD, synthetic dropout; UAS, upstream activating sequence; X-gal, 5-bromo-4-chloro-3-indo- suggesting a function in the early steps of microtubule assembly lyl-␤-D-galactopyranoside; HA, hemagglutinin; RAGE, rapid amplification of genomic DNA ends; BAC, bacterial artificial chromosome; ECL, enhanced chemiluminescence; GFP, green pathway (20–22). Our current data suggest that hMSH4 associates fluorescent protein. with VBP1 both in vitro and in vivo. 865

MATERIALS AND METHODS ing 0.13 mM methionine for efficient growth. Although the S. cerevisiae MET25-repressible promoter is activated in the absence of methionine, near Yeast Two-Hybrid Library Screening and Two- or Three-Hybrid Anal- complete repression requires 1 mM methionine (23). ysis. Human MutS homologue hMSH4 was used as a bait protein in the yeast Identification of a hMSH4sv and the hMSH4 Gene. Human hMSH4sv two-hybrid screening of a human ovary cDNA library (Clontech), in which we cDNA ORF sequence was obtained by performing PCR amplification with have identified VBP1 as an interacting protein partner for hMSH4. Specifi- human testis Marathon-Ready cDNA (Clontech Laboratories, Palo Alto, CA) cally, two-hybrid vector pAS2–1 containing full-length hMSH4 in frame with as a template using sense primer F-11 (5Ј-GGTTTGGGAGGATGCTGAG- GAL4-BD was used to perform two-hybrid screening of a human ovary cDNA GCCTGAGAT-3Ј) and antisense primer R2902 (5Ј-AATTTGGCAAATGT- ϫ 6 library in strain Y187. We have screened approximately 3.5 10 clones of TATTTTCCTTATCT-3Ј), corresponding to the 5Ј-UTR/ORF junction and the human ovary cDNA library on SD/-Ade-Leu-His-Trp medium for the 3Ј-UTR sequences, respectively (GenBank accession no. AF104243). The selection of positive protein-protein interactions by transcription activation of genomic clone harboring the human hMSH4 gene was obtained by a series of ADE2 and HIS3 reporter genes. All of the subsequent yeast two-hybrid complimentary approaches. First, one partial intron sequence of the human analyses were carried out by the use of the two-hybrid vectors, pAS2–1or hMSH4 gene was obtained by performing an anchored PCR amplification with pGBKT7 and pACT2 or pGADT7 (Matchmaker Two-Hybrid System; Clon- the RAGE template DNA as templates (8). To obtain a genomic clone tech), as well as S. cerevisiae strains Y187, Y190, or AH109. Yeast transfor- containing the hMSH4 gene, we used PCR-based screening of a “Down to the mants harboring both DNA-BD and activation domain constructs were se- Well” Human BAC Pool (Genome Systems, St. Louis, MO) with gene-specific lected on SD/-Leu-Trp medium. Positive protein-protein interactions were primers, exon-based F-11 and intron-based IN1R1 (5Ј-CTGTGAGGGACGT- ascertained by the transcription activation of highly inducible GAL1 UAS TATATGGGATACT-3Ј). One positive BAC clone, 265p14, was identified driving lacZ gene in the reporter host strains Y187 and Y190, as well as histidine prototrophy performed with Y190 or AH109 double transformants on and obtained from Genome Systems, Inc. Direct BAC DNA sequencing Ј SD/-Leu-Trp-His medium. ␤-galactosidase activities were qualitatively mon- indicated that the 265p14 clone contained only the 5 -end of the gene. There- itored by the blue color development with X-gal filter assays. fore, we next screened the RPCI 11 human male BAC library (Roswell Park The complex protein interactions among hMSH4, hMSH5, and VBP1 were Cancer Institute, Buffalo, NY) using hMSH4 cDNA as a probe, in which we analyzed with a three-hybrid approach (23), which was based on the expression identified an overlapping BAC clone, 262k24. To determine whether of an additional protein in its “native” form together with the expression of a hMSH4sv represented an alternative splicing product of the gene, we have DNA-BD fusion protein and an activation domain fusion protein within the partially sequenced the two overlapping hMSH4 BAC clones, 265p14 and same yeast cells. To generate constructs for the three-hybrid analysis, we have 262k24, within the coding regions and exon-intron junctions. cloned hMSH4, VBP1, and hMSH5 cDNAs into pBridge and pACT2 vectors Multiple Tissue cDNA Panel Analysis of hMSH4 and hMSH4sv Expres- (Clontech). Briefly, each pBridge construct is designed to express two of the sion in Human Tissues. Analysis of hMSH4 and hMSH4sv expression with three concerned genes as a BD-fusion and a HA-tagged protein. The pBridge Human Multiple Tissue cDNA panels (Clontech) was carried out by PCR with vector contains two cloning sites; the first cloning site is for the generation of primers F2273 (5Ј-TTGATGAACTTGGCAGAGGTACTA-3Ј) and R2685 a GAL4 DNA-BD fusion protein, and the second is used for generation of a (5Ј-AAGCCTAGTGGCTAGATGGTACAC-3Ј). Primers used in these analy- HA-tagged “nonfusion” protein under the control of the MET25-repressible ses were located on hMSH4 gene coding exons 17 and 20 and designed promoter. Positive protein-protein interactions were ascertained by simultane- to amplify 413-bp and 324-bp products representing hMSH4 and hMSH4sv ous transcription activation of both inducible GAL1 UAS driving HIS3 re- (Fig. 1). porter gene and GAL2 UAS driving ADE2 reporter gene in the reporter host Western Blot Analysis and Antibodies. SDS-PAGE was performed with strain AH109. Measurements of adenine and histidine prototrophy were per- 4–20% gradient acrylamide at 100 V for ϳ1.5 h. Proteins that had been formed with AH109 transformants on SD/-Ade-Leu-His-Trp medium contain- separated by SDS-PAGE were transferred to nitrocellulose membranes

Fig. 1. Human hMSH4 gene structure and multiple tissue cDNA panel-based analyses of human hMSH4 and hMSH4sv expression. A, hMSH4 gene organization. Black rectangles, portions of exons that encode ORF sequences; open rectangles,5Ј- and 3Ј-UTRs. ଙ, the hMSH4 exon 19 that is absent from hMSH4sv. B, analysis of human hMSH4 and hMSH4sv expression. PCR amplifications were performed with hMSH4 cDNA-specific primers, forward F2273 and reverse R2685 located on hMSH4 gene exons 17 and 20 (arrows in A), and designed to amplify 413-bp and 324-bp products representing hMSH4 and hMSH4sv, respectively. 866

(Schleicher and Schuell, Keene, NH) at 15 V for 2.5 h with a semidry transfer tained most of the highly conserved sequence motifs of MutS homo- apparatus (Bio-Rad Laboratories, Hercules, CA). Immunoreactive proteins logues except for the helix-turn-helix motif located within the COOH were detected with the ECL Western blotting system (Amersham Pharmacia terminus. hMSH4sv transcripts were expressed predominantly in hu- Biotech). Affinity-purified rabbit ␣-hMSH4 polyclonal antibody was gener- man testis with an estimated expression level reaching ϳ50% of the ated against a synthetic peptide corresponding to hMSH4 amino acid residues full-length transcript (Fig. 1). Very weak amplification signals for 920–936 (Y-K-E-D-F-P-R-T-E-Q-V-P-E-K-T-E-E), with the addition of a both hMSH4 and hMSH4sv were also observed from human brain, cysteine residue at the NH2 terminus of the peptide to mediate its coupling to keyhole limpet hemocyanin (Zymed Laboratories Inc., South San Francisco, pancreas, colon, and thymus (Fig. 1). These amplification products CA). Other antibodies used in this study include ␣-T7 tag monoclonal (No- could not have resulted from genomic DNA contamination because vagen, Madison, WI), ␣-HA monoclonal 12CA5 (Boehringer Mannheim), the two primers were located on hMSH4 coding exons 17 and 20 and ␣-Gal4 AD, and ␣-Gal4 BD monoclonal antibodies (Clontech). designed to amplify 413-bp and 324-bp products representing hMSH4 In Vitro Binding Assay. In vitro binding assay was used to validate the and hMSH4sv transcripts. interaction between hMSH4 and VBP1. To generate recombinant proteins To identify protein partners associated with hMSH4, full-length fused to either GST or His6-T7 tag, the hMSH4 and VBP1 coding sequences hMSH4 ORF was cloned into a Gal4 two-hybrid vector and was used were cloned in-frame into pGEX-6p (Pharmacia, Piscataway, NJ) and pTrc to screen a human ovary cDNA library. The hMSH4 protein bait (Invitrogen, San Diego, CA) vectors using a PCR-based approach. Native identified five individual clones, including four distinct ones, contain- Pyrococcus furiosus DNA polymerase (Stratagene, La Jolla, CA) was used in ing cDNA sequences with various lengths but all of them encoded all of the PCR amplifications. Fusion proteins were produced in either Esch- erichia coli BL21-Gold or BL21(DE3)-RIL strains (Stratagene). Overnight VBP1 protein. The longest VBP1 cDNA encoding full-length VBP1 induction of protein expression was carried out with 0.4 mM IPTG at 16°C. In protein was used for all of the subsequent analyses. Yeast two-hybrid vitro binding assay was performed essentially as described in detail elsewhere analyses demonstrated that hMSH4 specifically interacted with VBP1 (24). Briefly, the GST-VBP1 and GST control were purified with glutathion (Table 1). Considerable levels of ␤-galactosidase activities were ob- Sepharose 4B (Amersham Pharmacia Biotech). Immobilized GST or GST- tained when hMSH4-BD and VBP1-AD, or VBP1-BD and VBP1 on glutathion Sepharose 4B was then used to capture the second hMSH4-AD were coexpressed in Y187 and Y190, suggesting that recombinant protein His6-hMSH4. Similarly, the TALON Metal Affinity Res- hMSH4 associates with VBP1 (Table 1). On the contrary, there was ins (Clontech) were used to immobilize recombinant His6-VBP1 and a His6-tag no observed interaction between hMSH5 and VBP1 (Table 1). Inter- control protein, His -GFP and then were used to capture recombinant protein 6 estingly, the truncated protein encoded by hMSH4sv also interacted GST-hMSH4. Bound proteins were eluted with SDS sample buffer and sub- with VBP1 with strength equivalent to that between hMSH4 and jected to Western blot analysis for the presence of hMSH4 fusion proteins. Immunoprecipitation. Coimmunoprecipitation analysis of hMSH4 and VBP1, suggesting that the protein encoded by the splicing variant VBP1 interaction was carried out by the use of bacterial expressed recombi- could be functional. However, the truncated hMSH4sv protein could nant fusion proteins. pGEX-6p and pET-28a (Novagen) vectors were used to not interact with hMSH5, indicating that the helix-turn-helix motif generate expression constructs for fusion proteins GST-hMSH4 and His6- located at the COOH terminus of hMSH4 is critical for interaction VBP1 containing the T7 tag epitope. Coexpression of recombinant proteins with hMSH5 (Table 1). A separate reporter gene HIS3 in strain Y190 from pGEX-6p- and pET-28a-based constructs was carried out in E. coli or AH109 was also used to validate the observed protein interactions. BL21(DE3)-RIL with the addition of 0.4 mM IPTG to induce protein expres- As shown in Table 1, histidine prototrophy phenotypes were ascer- sion at 16°C overnight. Soluble fractions of whole-cell lysate were prepared in tained from Y190 or AH109 double transformants harboring the same ϫ ϫ 1 PBS containing 1 complete EDTA-free protease inhibitor cocktail pairs of two-hybrid constructs that could lead to LacZ gene activation. (Roche, Mannheim, Germany). Cell lysates were then incubated overnight The observed protein interactions were further substantiated when we with 5 ␮g of affinity-purified ␣-hMSH4 polyclonal or 2.5 ␮g of monoclonal ␣-T7 tag antibody at 4°C. Immunoprecipitates were captured with 75 ␮lof 50% slurry of BSA-saturated Protein A/G PLUS-Agarose (Santa Cruz Bio- technology). Table 1 Two-hybrid analysis of hMSH4 and VBP1 interactions Colocalizaton of hMSH4 and VBP1 in Mammalian Cells. Subcellular ␤-Galactosidase activity was determined with the X-gal filter assay, and the strength colocalization of hMSH4 and VBP1 was carried out by the use of fluorescent of color development was scored in reference to that of the positive control (pVA3-1 and ϩϩϩ fusion proteins. To generate GFP-hMSH4 and VBP1-DsRed2 proteins, pTD1-1) with three plus signs ( ) representing the strongest interaction. pAS2-1 and pACT2 are two-hybrid vectors containing Gal4 DNA-BD and AD, respectively. pVA3-1 hMSH4 and VBP1 cDNAs were subcloned into pEGFP and pDsRed2 (Clon- and pTD1-1 are two-hybrid constructs expressing Gal4-BD-p53 and Gal4-AD-T antigen tech) vectors, respectively. The resulting expression constructs were trans- fusion proteins, respectively. Histidine prototrophy was measured on SD/-Leu-Trp-His/ fected into MSC-1 cells (25, 26), and transfected cells were maintained in 3-AT medium, and the growth of double transformants was also scored with reference to that of the T-antigen and p53 controls. DMEM supplemented with 10% newborn calf serum at 37°C and 5% CO2 for 48 h. Confocal images of subcellular distributions of fluorescent hMSH4 and ␤-Galactosidase VBP1 fusion proteins were acquired by the use of Nikon Eclipse TE300 Two-hybrid constructs activity Histidine prototrophy hooked to Bio-Rad MRC 1024 laser confocal microscope with LaserSharp 3.0 DNA-BD AD Y187 Y190 (Y190 or AH109) software (Bio-Rad Laboratories). hMSH4 pACT2 (AD) ϪϪ Ϫ hMSH4sv pACT2 (AD) ϪϪ Ϫ RESULTS VBP1 pACT2 (AD) ϪϪ Ϫ pAS2-1 (BD) hMSH4 ϪϪ Ϫ pAS2-1 (BD) hMSH4sv ϪϪ Ϫ As a step toward to the identification of hMSH4 interacting protein pAS2-1 (BD) VBP1 ϪϪ Ϫ partners, we have isolated hMSH4 ORF sequence by PCR from a hMSH4 VBP1 ϩϩϩ ϩϩϩ ϩϩϩ human testis cDNA preparation (9). Sequence analysis of cloned VBP1 hMSH4 ϩϩϩ ϩϩϩ ϩϩϩ hMSH4sv VBP1 ϩϩϩ ϩϩϩ ϩϩϩ hMSH4 ORFs indicated that, in addition to the full-length hMSH4 VBP1 hMSH4sv ϩϩϩ ϩϩϩ ϩϩ ORF, we have also identified a hMSH4sv. Sequence information hMSH4sv hMSH5 ϪϪ Ϫ hMSH5 hMSH4sv ϪϪ Ϫ generated from the two overlapping hMSH4 BAC clones, 265p14 and hMSH4 hMSH5 ϩϩϩ ϩϩϩ ϩϩϩ 262k24, indicated that hMSH4sv lacked 89-bp between nucleotide hMSH5 hMSH4 ϩϩϩ ϩϩϩ ϩϩϩ positions 2530 and 2620 corresponding to the entire exon 19. This VBP1 hMSH5 ϪϪ Ϫ hMSH5 VBP1 ϪϪ Ϫ alternative splicing resulted in a frameshift of 7 amino acids followed pVA3-1 (p53) pTD1-1 (T-Antigen) ϩϩϩ ϩϩϩ ϩϩϩ by a stop codon at nucleotides 2640–2642 in exon 20 (Fig. 1). The pVA3-1 (p53) pACT2 ϪϪ Ϫ 850-amino acid-truncated protein encoded by hMSH4sv ORF con- pAS2-1 pTD1-1 (T-Antigen) ϪϪ Ϫ 867

Fig. 2. In vitro binding assay for determination of hMSH4 and VBP1 interaction. Recombinant hMSH4 and VBP1 proteins were produced as either GST or 6ϫ His tag fusion proteins. A, the GST-VBP1 and GST control were captured with Glutathion Sepha- rose 4B. Immobilized GST or GST-VBP1 was then used to capture recombinant protein His6-hMSH4. B, similarly, the TALON Metal Affinity Resins was used to immobilize recombinant His6-VBP1 and control fusion protein His6-GFP, and then used to capture GST-hMSH4. Western blot analysis was performed to detect the presence of hMSH4 proteins with an affinity-purified ␣-hMSH4 polyclonal antibody. C, analysis of hMSH4 and VBP1 interaction through coimmunoprecipitation. Coexpression of recombinant GST-hMSH4 and His6-VBP1 proteins was carried out in E. coli BL21 (DE3)-RIL. Lysates were collected from bacterial cells harboring different pairs of expression constructs, which included: (1) hMSH4/ pGEX-6p and VBP1/pET-28a constructs; (2) hMSH4/pGEX-6p constructs and pET-28a vector; (3) pGEX-6p vector and VBP1/pET-28a construct. A standard immunoprecipitation procedure was carried out with either ␣-hMSH4 or ␣-T7 antibodies (see “Materials and Methods” for details). Equal volumes of the immunoprecipitates, along with purified GST- hMSH4 and His6-VBP1 fusion proteins, were re- solved on 4–20% SDS-PAGE, transferred onto nitrocellulose membranes, and subjected to Western blot analysis for the presence of hMSH4 and VBP1 proteins with either an affinity-purified ␣-hMSH4 polyclonal or a monoclonal ␣-T7 antibody, respectively. kDa, molecular weight (Mr) in thousands.

failed to observe LacZ and HIS3 gene activation in yeast double capable of immunoprecipitating GST-hMSH4 and His6-T7-VBP1 fu- transformants expressing only one of the two testing proteins in either sion proteins, respectively (Fig. 2C, Lanes 1 and 5). Monoclonal ␣-T7 BD or AD fusion form (Table 1). tag antibody against VBP1 fusion protein could precipitate hMSH4 An in vitro binding assay was then performed to investigate the only when VBP1 was present (Fig. 2C, Lanes 2 and 4). Furthermore, interaction between hMSH4 and VBP1. To generate GST-fusion and immunoprecipitation of VBP1 with affinity-purified ␣-hMSH4 anti-

His6-T7 tagged proteins, the hMSH4 and VBP1 coding sequences body was entirely dependent on the expression of hMSH4 protein were cloned in-frame into pGEX-6p and pTrcHis vectors and intro- (Fig. 2C, Lanes 6 and 7); His6-T7-VBP1 was detected only when duced into BL21(DE3)-RIL cells for the generation of recombinant lysate containing both hMSH4 and VBP1 was used but not with the proteins. The GST-VBP1 and GST control proteins were purified control lysate expressing VBP1 and GST (Fig. 2C; Lanes 6 and 7). from soluble fraction and immobilized onto glutathion-Sepharose Taken together, these results suggest that hMSH4 specifically inter- beads. Immobilized GST-VBP1 or GST proteins on glutathion-Sepha- acts with VBP1 in solution. rose beads were then incubated with bacterial lysates containing We next set out to identify hMSH4 domains responsible for inter- recombinant His6-T7-hMSH4 fusion protein. Proteins that retained on action with VBP1 using the two-hybrid analysis. As shown in Fig. 3, glutathion-Sepharose beads were eluted in sample buffer and sepa- a series of deletion hMSH4 mutants were generated in GAL4-based rated by SDS-PAGE, which was followed by transferring onto nitro- BD or AD vectors and were used to perform two-hybrid analyses. cellulose membranes and immunoblotting with affinity-purified Positive protein interactions were ascertained by the transcription ␣-hMSH4 antibody. Apparently, GST-VBP1 fusion protein, but not activation of highly inducible GAL1 UAS driving lacZ gene in the GST alone, could result in the retention of hMSH4 protein (Fig. 2A). reporter strains and were determined qualitatively with the X-gal filter

Similarly, the immobilized recombinant His6-T7-VBP1 protein on assay. The results of these analyses indicated that both NH2 and TALON metal affinity resins could capture GST-hMSH4 protein, COOH termini of hMSH4 were not required for interaction with whereas immobilized control fusion protein His6-GFP did not display VBP1 (Fig. 3). In particular, hMSH4 deletion mutant, lacking the first the same property (Fig. 2B). Consistent with the results of the two- 147 amino acid residues, maintained the ability to interact with VBP1; hybrid analysis, the in vitro binding assay demonstrated that recom- however, any further deletion from the NH2 terminal abolished that binant VBP1 protein specifically interact with recombinant hMSH4. interaction (Fig. 3). This observation indicated that the first 147 amino To address whether this interaction could occur in solution between acid residues of hMSH4 were not involved in mediating protein hMSH4 and VBP1, immunoprecipitation experiments were performed interaction with VBP1, coupling the fact that this portion of the amino with the soluble fraction of the bacterial lysates containing both acid sequence is highly divergent between human and mouse MSH4 hMSH4 and VBP1 fusion proteins. Recombinant GST-hMSH4 and (10). Although hMSH4 amino acid residues 844–936 containing the

His6-T7-VBP1 fusion proteins were coexpressed in BL21(DE3)-RIL helix-turn-helix motif were not required for interaction with VBP1, as host cells. Soluble fractions of cell lysates were prepared from cells suggested by the observed interaction between hMSH4sv and VBP1, expressing both fusion proteins and cells expressing either of the two, further deletion of the region connecting the putative nucleotide- followed by incubation with ␣-hMSH4 or monoclonal ␣-T7 tag binding motif and helix-turn-helix motif abolished the interaction antibodies. Immunoprecipitants were analyzed by immunoblotting (Fig. 3). In addition, all of the other truncated mutations, containing performed with either ␣-hMSH4 or monoclonal ␣-T7 tag antibodies. different lengths of hMSH4 peptide sequences, would not interact The results of these experiments demonstrated that the affinity-puri- with VBP1 (Fig. 3). Therefore, it is conceivable that most of the fied polyclonal ␣-hMSH4 and monoclonal ␣-T7 tag antibodies were hMSH4 peptide sequence is required for mediating protein interac- 868

tion in the same biological pathway (9–13). Therefore, the observation that hMSH4 could interact with VBP1 raised interesting ques- tions regarding whether VBP1 would compete with hMSH5 for the binding of hMSH4. We next attempted to investigate the interplay of hMSH4, VBP1, and hMSH5 proteins with regard to the potential formation of protein complex. We have adapted the three-hybrid approach to determine whether there was a cooperative or competitive interaction among these three proteins. As indicated in Fig. 5A, the activation of both HIS3 and ADE2 reporter genes, as determined by the growth of yeast transformants on SD/-Ade-Leu-His-Trp medium, were obtained only when BD-hMSH4 and AD-hMSH5 or BD- hMSH4 and AD-VBP1 were coexpressed in host strain AH109 (yeast transformants no. 13 and no. 15). However, the activation of these reporter genes was almost completely inhibited when HA-VBP1 or HA-hMSH5 was coexpressed in the corresponding yeast transformants (no. 14 and no. 16). These observations suggested that VBP1 might compete with hMSH5 for the binding of hMSH4. This view was also supported by the lack of reporter gene activation in yeast transformant no. 18 (coexpressing BD-hMSH5, AD-VBP1, and HA- hMSH4) and transformant no. 20 (coexpressing BD-VBP1, AD- hMSH5, and HA-hMSH4), suggesting that hMSH4 could not interact with VBP1 and hMSH5 concomitantly. Similar experiments performed with a separate reporter strain Y187 provided identical results (data not shown). The expression of relevant fusion proteins in the concerned AH109 transformants was validated with Western blot analysis, so that the failure in reporter gene activation was not attrib- utable to the absence of protein expression (Fig. 5B). These observations were further supported when we failed to observe reporter gene activations in AH109 double transformants coexpressing only one of the three testing proteins in either AD or BD fusion form (Fig. 5A, transformants no. 1 to no. 6). Furthermore, AH109 transformants Fig. 3. Identification of VBP1-interacting domains on hMSH4. The 936-amino acid coexpressing any combination of two proteins in BD-fusion and hMSH4, 843-amino acid hMSH4sv, and 197-amino acid VBP1, as well as a series of native forms could not grow on SD/-Ade-Leu-His-Trp medium (Fig. hMSH4 deletion mutants were schematically depicted as either BD or AD fusion proteins. The numbers with the arrows, the amino acid positions within that protein. Single solid 5A, transformants no. 7 to no. 12). In contrast, all of the AH109 lines, the portions of peptide sequences that have been removed. Gray boxes, the double transformants grew efficiently on SD/-Leu-Trp medium (Fig. ATP-binding consensus regions, as described by New et al. (33), and the helix-turn-helix motif defined by Ross-Macdonald and Roeder (6). Analysis of protein interactions was 5A; bottom panel). These results indicated that hMSH4-hMSH5 het- carried out with Matchmaker Two-Hybrid System and S. cerevisiae strains Y187 and erodimer could be disrupted by the overexpression of VBP1 protein. Y190. Positive protein interactions were ascertained by the transcription activation of highly inducible GAL1 UAS driving lacZ gene in the reporter strains. ␤-Galactosidase only the BD-hMSH4 DISCUSSION ,ء .activities were determined qualitatively with the X-gal filter assay 148–936 configuration showed positive interaction with AD-VBP1. In the present study, we aimed to identify human hMSH4-associ- ating protein partners and to understand their potential effects on the tions with VBP1. Alternatively, it is also possible that the VBP1- formation of hMSH4 and hMSH5 heterocomplex. With the full-length interacting region is composed of multiple noncontinuous regions of hMSH4 as bait, we have identified VBP1 as a putative protein-binding hMSH4 protein. partner for hMSH4. The in vitro binding assay demonstrated a direct To demonstrate that the association of hMSH4 and VBP1 proteins interaction between hMSH4 and VBP1 proteins, which was further could occur in vivo, we have attempted to determine whether hMSH4 confirmed by immunoprecipitation experiments (Fig. 2). Moreover, colocalizes with VBP1 in cultured mammalian cells. To this end, hMSH4 associated with VBP1 and formed cytoplasmic foci in MSC-1 MSC-1 cells were transfected with either an individual construct or a cells (Fig. 4). We have also identified and characterized a hMSH4sv mixture of both expression constructs encoding GFP-tagged hMSH4 that encoded a truncated protein lacking the conserved COOH termi- or DsRed2-tagged VBP1 proteins. As shown in Fig. 4, VBP1 was nal helix-turn-helix motif. Although the truncate hMSH4sv protein predominately localized in the cytoplasm, particularly displayed as could not interact with hMSH5, it interacted with VBP1 at approxi- perinuclear foci. The observed VBP1 subcellular distribution pattern mately equivalent strength as the full-length protein (Table 1), sug- was nearly identical, as reported previously (18). Although hMSH4 gesting that hMSH4sv might represent a separation-of-function alter- distributed diffusely in the cytoplasm without concomitant expression native. This observation leads to the possibility that hMSH4sv protein of VBP1 (Fig. 4A), it appeared that hMSH4 fusion protein could might be functional and, therefore, involved in the same pathway of redistribute and accumulate prominently within VBP1 foci in cells VBP1. In addition, the inability of hMSH4sv to interact with hMSH5 expressing both proteins (Fig. 4C). Together, these data suggested that indicates that the conserved helix-turn-helix motif of hMSH4 is crit- hMSH4 and VBP1 proteins resided predominantly in the cytoplasm, ical for protein interaction with hMSH5. It is conceivable that the and VBP1 could trigger the formation of hMSH4-VBP1 foci. human hMSH4 might encode at least two functional distinct proteins; It is known that mammalian MSH4 forms heterocomplex with the full-length protein could interact with both hMSH5 and VBP1, MSH5 and mice lacking either functional Msh4 or Msh5 displayed whereas the alternative form interacts only with VBP1. Both hMSH4 similar meiotic phenotypes, suggesting that these genes might func- and hMSH4sv transcripts were expressed predominantly in human 869

Fig. 4. Subcellular colocalizaton of hMSH4 and VBP1 in MSC-1 cells. Confocal microscopic analysis of hMSH4 and VBP1 localization was carried out with GFP or DsRed2 fluorescent fusion proteins, respectively. A, MSC-1 cells were transfected with hMSH4/pEGFP plasmid DNA. B, MSC-1 cells were transfected with VBP1/pDsRed2 construct. C, MSC-1 cells were cotransfected with hMSH4/pEGFP and VBP1/pDsRed2 plasmids. Confocal images were acquired at different magnifications for cells expressing individual proteins (A, B) and cells coexpressing both fusion proteins (C). testis; the level of hMSH4sv expression was ϳ50% of that of the two protein complexes and the question as to when they become hMSH4 (Fig. 1) critical during the meiotic process remain to be clarified. Another Homologues of the human VBP1 protein were highly conserved in interesting question is whether the expression of hMSH4 and mammals and yeast (18–20). It has been shown that VBP1 played an hMSH4sv proteins is subject to coordinate regulation. Although the essential role in promoting the formation and assembly of functional stoichiometry of the functional protein complex consisting of hMSH4 ␣-, ␤-, and ␥-tubulin; thus, it functions in the early steps of the and VBP1 has not yet been directly evaluated, several lines of evi- microtubule assembly pathway (20–22). It is known that microtubules dence indicate that the formation of hMSH4-VBP1 multimeric func- play an important role in numerous cellular processes including chro- tional complex, such as a tetramer, is theoretically possible. It has mosome segregation in mitosis and meiosis. Thus, the observed been shown that both yeast and human MSH4 was able to form interaction between VBP1 and hMSH4 (or hMSH4sv) proteins sug- homo-oligomer structures (9, 11), and the human VBP1 protein could gested that hMSH4 might possess multiple functions during the mei- mediate strong homotypic interactions in a yeast two-hybrid analysis otic process; besides its role in meiotic recombination, hMSH4 might (data not shown). be directly or indirectly involved in the manipulation of microtubule MSH4 and MSH5 were not required for the completion of meiosis assembly to maintain proper chromosome positioning during meiotic in both yeast and Caenorhabditis elegans (5, 6, 28, 29); conversely, processes. It has been shown that disruption of the yeast VBP1 the successful completion of meiosis in mice required functional homologous gene resulted in the generation of malfunctioning spores Msh4 and Msh5 genes (14–16). In addition to meiotic defects, inac- and, therefore, leads to germination defects (27). Yeast VBP1 homo- tivation of either Msh4 or Msh5 in mice resulted in testicular and logue was not essential for vegetative growth; however, deletion ovarian degeneration; inactivation of these two genes had more severe mutants lacking the functional gene displayed a slow growth pheno- effects on ovary developments because complete ovarian dysgenesis type (27). Previous studies performed with mammalian MSH4 and in Msh4- and Msh5-deficient mice by adulthood was observed appar- MSH5 indicated that these two proteins could form a heterocomplex, ently as a result of massive apoptosis (14–16). These findings are and targeted mutation of either Msh4 or Msh5 in mice caused particularly important in light of the fact that no other genes known to pachytene arrest because of disrupted chromosome pairing and, there- be involved in meiotic processes associate with this profound pheno- fore, female and male sterility (9, 10, 12–16). Interestingly, results of type; coupled with the fact that yeast VBP1 mutants display a growth our current study have suggested that VBP1 could compete with defect (27), the current data are consistent with the view that the hMSH5 for the binding of hMSH4, raising the possibility that hMSH4 hMSH4-VBP1 interaction might be involved in cellular processes might play multiple functional roles through interactions with differ- critical for ovarian development. To this end, it would be of interest ent protein partners. It appeared, however, that the existence of to know whether hMSH4-VBP1 and hMSH4sv-VBP1 complexes hMSH4sv protein might represent a mechanism to alleviate the com- would possess similar functional properties and whether there is any petitive tension between VBP1 and hMSH5 to form protein complex spatial and temporal control for the formation of different heterocom- with hMSH4. Of course, the specific functions associated with these plexes among these three proteins. 870

Fig. 5. Analysis of complex protein interactions among hMSH4, VBP1, and hMSH5. All of the relevant constructs used in the three-hybrid analysis were generated with pBridge and pACT2 vectors, and relevant constructs were transformed into S. cerevisiae reporter strain AH109. Positive protein-protein interactions were ascertained by simultaneous transcription activation of inducible GAL1 UAS driving HIS3 reporter gene and GAL2 UAS driving ADE2 reporter gene in the reporter host strain AH109, which displayed adenine (Ade) and histidine (His) prototrophy phenotypes as measured on SD/-Ade-Leu-His-Trp medium. A,20 AH109 double transformants, expressing one, two, or three fusion proteins representing different combina- tions of hMSH4, VBP1, and hMSH5 in BD, AD, or HA fusion forms, were spotted at various dilutions on selection medium SD/-Ade-Leu-His-Trp (top panel), and with single dilution on SD/-Leu-Trp (bottom panel). AH109 transformants no. 1 to no. 12, neg- ative controls for the analysis. B, validation of expression of relevant fusion proteins in represent- ative AH109 double transformants by Western blot analysis performed with ␣-hMSH4, ␣-HA, ␣-Gal4 AD, and ␣-Gal4 BD antibodies. kDa, molecular weight (Mr) in thousands.

Our present study also indicated that hMSH5 was predominately processes; it is plausible that the cellular effect of hMSH4-VBP1 presented in the cytoplasm with subcellular localization patterns that interaction is on the manipulation of microtubule assembly during were similar to those of hMSH4 (data not shown). Hence, the trans- meiotic chromosome segregation and, therefore, plays a role in the location of these proteins into the nucleus must be regulated by a maintenance of chromosomal stability. Meiotic nondisjunction is the yet-to-be-identified mechanism (14–16). One important aspect in the major cause for aneuploidy, which leads to physical abnormalities and functional analysis of these protein complexes is to determine whether metal retardation in humans (32). One may expect that disruption of they have functions beyond the scope of meiosis. It is known that protein interactions because of mutations of these protein factors can mammalian VBP1 is ubiquitously expressed in adult tissues (18, 19, cause meiotic defects in humans. 30). In contrast to the meiotic-restricted expression pattern of mammalian MSH4, transcripts, and in some cases proteins of human ACKNOWLEDGMENTS hMSH5, have been identified in almost all of the tissues examined (Refs. 8, 12, 13; data not shown). Consistent with a previous report We thank Dr. Norman Doggett, Dr. Cliff Han, Dr. Hong Cao, Tai-Hsien Lee, that yeast MSH5 is also involved in mitotic processes (31), these Robert Adair, and Dr. DongKee Jeong for their helpful advice and assistance. observations suggest that hMSH5 might play functional roles in human nonmeiotic tissues either in the absence or in the presence of REFERENCES hMSH4. In summary, our present study suggests that VBP1 may act 1. Buermeyer, A. B., Deschenes, S. M., Baker, S. M., and Liskay, R. M. Mammalian as a protein partner for both hMSH4 and hMSH4sv during meiotic DNA mismatch repair. Annu. Rev. Genet., 33: 533–564, 1999. 871

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Chengtao Her, Xiling Wu, Michael D. Griswold, et al.

Cancer Res 2003;63:865-872.

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