[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, re- sulting 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 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. hMSH4 AND VBP1 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