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• W1967110156 endingPage "24537" @default.
• W1967110156 startingPage "24530" @default.
• W1967110156 abstract "We have identified a novel DNA helicase in humans that belongs to members of the superfamily I helicase and found that it contains a well conserved F-box motif at its N terminus. We have named the enzyme hFBH1 (human F-box DNAhelicase 1). Recombinant hFBH1, containing glutathione S-transferase at the N terminus, was expressed in Sf9 cells and purified. In this report, we show that hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that displace duplex DNA in the 3′ to 5′ direction. The hFBH1 enzyme interacted with human SKP1 and formed an SCF (SKP1/Cullin/F-box) complex together with human Cullin and ROC1. In addition, the SCF complex containing hFBH1 as an F-box protein displayed ubiquitin ligase activity. We demonstrate that hFBH1 is the first F-box protein that possesses intrinsic enzyme activity. The potential role of the F-box motif and the helicase activity of the enzyme are discussed with regard to regulation of DNA metabolism. We have identified a novel DNA helicase in humans that belongs to members of the superfamily I helicase and found that it contains a well conserved F-box motif at its N terminus. We have named the enzyme hFBH1 (human F-box DNAhelicase 1). Recombinant hFBH1, containing glutathione S-transferase at the N terminus, was expressed in Sf9 cells and purified. In this report, we show that hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that displace duplex DNA in the 3′ to 5′ direction. The hFBH1 enzyme interacted with human SKP1 and formed an SCF (SKP1/Cullin/F-box) complex together with human Cullin and ROC1. In addition, the SCF complex containing hFBH1 as an F-box protein displayed ubiquitin ligase activity. We demonstrate that hFBH1 is the first F-box protein that possesses intrinsic enzyme activity. The potential role of the F-box motif and the helicase activity of the enzyme are discussed with regard to regulation of DNA metabolism. ubiquitin-protein isopeptide ligase amino acid ubiquitin ubiquitin-activating enzyme ubiquitin carrier protein glutathione S-transferase expressed sequence tag rapid amplification of cDNA ends hemagglutinin DNA helicases are ubiquitous enzymes that play essential roles in various DNA transactions involved in replication, repair, and recombination (1Matson S.W. Kaiser-Rogers K.A. Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (335) Google Scholar, 2Matson S.W. Bean D.W. George J.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (269) Google Scholar) and have been implicated in a number of human genetic disorders (3Ellis N.A. Curr. Opin. Genet. Dev. 1997; 7: 354-363Crossref PubMed Scopus (115) Google Scholar, 4Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Crossref PubMed Scopus (188) Google Scholar). A trademark of these enzymes is a set of well conserved amino acid sequences termed “helicase motifs” (5Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1022) Google Scholar, 6Hall M.C. Matson S.W. Mol. Microbiol. 1999; 34: 867-877Crossref PubMed Scopus (269) Google Scholar). Helicases are generally believed to generate single-stranded DNA by catalyzing the melting of stable helical DNA structures utilizing energy derived from the hydrolysis of nucleoside triphosphates. Analysis of the genome of Saccharomyces cerevisiae indicates that there are 134 open reading frames containing with helicase-like features that represent ∼2% of its genome (7Shiratori A. Shibata T. Arisawa M. Hanaoka F. Murakami Y. Eki T. Yeast. 1999; 15: 219-253Crossref PubMed Scopus (93) Google Scholar). The presence of such a large number of helicases or helicase-like proteins in cells, many of unknown function in vivo, most likely reflects a complexity of nucleic acid metabolic reactions and the distinct structural template requirements for a given helicase. Alternatively, many helicases may play roles that functionally overlap, making it difficult to determine a specific role for a given helicase. For example, each mutant cell of the two yeast helicases, Sgs1 and Srs2, grows with wild-type kinetics, but the combination of an sgs1 mutation with loss of the helicase srs2 resulted in a severe growth defect (8Lee S.K. Johnson R.E., Yu, S.L. Prakash L. Prakash S. Science. 1999; 286: 2339-2342Crossref PubMed Scopus (126) Google Scholar, 9McVey M. Kaeberlein M. Tissenbaum H.A. Guarente L. Genetics. 2001; 157: 1531-1542Crossref PubMed Google Scholar). A majority of sgs1 and srs2 mutant cells stops dividing stochastically as large budded DNA (9McVey M. Kaeberlein M. Tissenbaum H.A. Guarente L. Genetics. 2001; 157: 1531-1542Crossref PubMed Google Scholar). Such double mutant cells are unable to replicate DNA at restrictive temperature and unable to efficiently transcribe rDNA (8Lee S.K. Johnson R.E., Yu, S.L. Prakash L. Prakash S. Science. 1999; 286: 2339-2342Crossref PubMed Scopus (126) Google Scholar). This suggests that these two helicases provide a redundant but essential activity for DNA replication and rDNA transcription. Despite well conserved motifs specific to helicase family proteins, there are several proteins for which no helicase activity has been demonstrated, indicating that helicase motifs cannot, without additional information, be used to define a protein as a helicase. For example, the chromatin-remodeling factor SWI2/SNF2 containing these motifs lacks helicase activity, although its ability to hydrolyze ATP is stimulated by DNA (10Richmond E. Peterson C.L. Nucleic Acids Res. 1996; 24: 3685-3692Crossref PubMed Scopus (110) Google Scholar, 11Travers A. Cell. 1999; 96: 311-314Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), suggesting that ATP hydrolysis provides the energy required to alter protein-DNA structure rather than duplex DNA or RNA structure. This suggests that proteins with helicase motifs may have functions that do not involve unwinding of nucleic acids.As a continued effort to understand the role ofSchizosaccharomyces pombe DNA helicase I that we reported previously (12Park J.S. Choi E. Lee S.H. Lee C. Seo Y.S. J. Biol. Chem. 1997; 272: 18910-18919Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), we identified a human homolog of this fission yeast enzyme and named it hFBH1 (humanF-box DNA helicase 1) because it contains a well conserved functional F-box motif (13Bai C. Sen P. Hofmann K., Ma, L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). In this report, we present data that hFBH1 is not only an ATPase/DNA helicase but also is an F-box protein that can form an SCF (SKP1-CUL1 (Cdc53)-Rbx1-F-box protein) complex with human SKP1 and CUL1. SCF complexes constitute a new class of E31 ligase that plays important roles in cell cycle regulation and signal transduction by catalyzing ubiquitin-mediated proteolysis (14Peters J.M. Curr. Opin. Cell Biol. 1998; 10: 759-768Crossref PubMed Scopus (224) Google Scholar, 15Craig K.L. Tyers M. Prog. Biophys. Mol. Biol. 1999; 72: 299-328Crossref PubMed Scopus (236) Google Scholar, 16Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 21: 1180-1182Abstract Full Text Full Text PDF Scopus (305) Google Scholar, 17Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 18Tyers M. Jorgensen P. Curr. Opin. Genet. Dev. 2000; 10: 54-64Crossref PubMed Scopus (268) Google Scholar). The substrate specificity of an SCF complex is governed by the interchangeable F-box protein subunit, which recruits a specific set of substrates for ubiquitination to the SCF core complex composed of SKP1, Cdc53, ROC1, and the E2 enzyme Cdc34. The polyubiquitinated proteins are rapidly captured by the 26 S proteasome, an abundant, self-compartmentalized protease particle (19Baumeister W. Walz J. Zuhl F. Seemuller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). To date, hFBH1 is the first example of an F-box protein that contains intrinsic enzymatic activity, suggesting that a helicase can play a role in a certain aspect of DNA metabolism that requires ubiquitin-dependent proteolysis. The potential biological role of this interesting human DNA helicase will be discussed.DISCUSSIONIn this report, we demonstrate that hFBH1 is a DNA helicase that translocates in the 3′ to 5′ direction and interacts with SKP1 in a manner that requires a functional F-box motif. Besides, hFBH1 interacted with CUL1/ROC1 and formed a SCF complex that contained all known four subunits (SKP1, hFBH1, CUL1, and ROC1). Moreover, SCFhFBH1 displayed ubiquitin ligase activity in a F-box motif-dependent fashion. The polyubiquitination activity of the SCFhFBH1 complex was dependent upon functional E1 and E2 enzymes, demonstrating that the complex interacts with the ubiquitination machinery. Our study showed that hFBH1 is the first example of an F-box protein that harbors an intrinsic enzymatic activity, catalyzing a helicase reaction. In addition, hFBH1 is the first F-box protein implicated in nucleic acid metabolism. Therefore, our findings raise the possibility that the role of hFBH1 is most likely to recruit a target protein, most likely involved in some aspect of DNA metabolism, to the protein degradation pathway. Thus, a protein involved in DNA metabolism would be marked by hFBH1 for degradation via the highly regulated ubiquitin degradation pathway.As an initial attempt to evaluate the role of the F-box motif of the enzyme in this regard, we decided to identify a protein(s) that interacts specifically with hFBH1 using the yeast two-hybrid screen. This screen resulted in the isolation of human MAT1 (data not shown), which is a regulatory subunit of the Cdk-activating kinase (29Yee A. Nichols M.A., Wu, L. Hall F.L. Kobayashi R. Xiong Y. Cancer Res. 1995; 55: 6058-6062PubMed Google Scholar, 30Yee A., Wu, L. Liu L. Kobayashi R. Xiong Y. Hall F.L. J. Biol. Chem. 1996; 271: 471-477Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). It is unlikely that MAT1 is a direct substrate of SCFhFBH1 E3 ligase activity because a substrate phosphorylation is a prerequisite step prior to recognition by an F-box protein (31Elsasser S. Chi Y. Yang P. Campbell J.L. Mol. Biol. Cell. 1999; 10: 3263-3277Crossref PubMed Scopus (134) Google Scholar, 32Deshaies R.J. Curr. Opin. Genet. Dev. 1997; 7: 7-16Crossref PubMed Scopus (82) Google Scholar). MAT1 in Cdk-activating kinase may play a role leading to the phosphorylation of a substrate in proximity with hFBH1, thereby facilitating the subsequent ubiquitination of the substrate by the SCFhFBH1complex. In support of this possibility, we failed to detect any ubiquitination of MAT1 (data not shown). These findings raise the possibility that MAT1 may play a direct role in the phosphorylation of hFBH1 instead of acting as a substrate for the E3 ligase activity of the SCFhFBH1 complex. Therefore, the level of hFBH1 itself is likely to be regulated by posttranslational modifications that involve both Cdk-activating kinase and E3 ligase activities. Because it has been reported that F-box proteins can be degraded autocatalytically in a ubiquitination-dependent fashion (33Galan J.M. Peter M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9124-9129Crossref PubMed Scopus (226) Google Scholar), the F-box motif of hFBH1 may regulate the level of hFBH1 through auto-ubiquitination during cell cycle progression. However, we were not able to demonstrate this, because the endogenous levels of hFBH1 were too low to be detected with the antibodies that we have (data not shown). We also explored whether hFBH1 is ubiquitinated in vivo. Although the hFBH1 was indeed ubiquitinated in vivo (data not shown), the efficiency of hFBH1 ubiquitination was independent of the intact F-box motif, suggesting that ubiquitination occurred in trans by some other ubiquitinating activity. Reconstitution of the SCF complex containing hFBH1 with the purified proteins will help to address this question.Although the biological function in vivo of hFBH1 is unclear at present, mutational studies of the S. pombe fdh1 + gene (encoding the S. pombe homolog of hFBH1) have provided some clues. When the S. pombe fdh1 + gene was deleted, the cells showed elongated cell morphology and uneven distribution of chromosomes in dividing cells. 2S. H. Lee and Y. S. Seo, unpublished observation. In addition, deletion of either the F-box motif or a single amino acid change in the ATP-binding motif resulted in phenotypes similar to those of the null mutation.2 These observations suggest that both the F-box and ATPase/helicase activity of hFBH1 are required for the physiological function of the enzyme. Currently, both genetic and biochemical studies are underway using an S. pombe model system to delineate the precise biological role of hFBH1. DNA helicases are ubiquitous enzymes that play essential roles in various DNA transactions involved in replication, repair, and recombination (1Matson S.W. Kaiser-Rogers K.A. Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (335) Google Scholar, 2Matson S.W. Bean D.W. George J.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (269) Google Scholar) and have been implicated in a number of human genetic disorders (3Ellis N.A. Curr. Opin. Genet. Dev. 1997; 7: 354-363Crossref PubMed Scopus (115) Google Scholar, 4Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Crossref PubMed Scopus (188) Google Scholar). A trademark of these enzymes is a set of well conserved amino acid sequences termed “helicase motifs” (5Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1022) Google Scholar, 6Hall M.C. Matson S.W. Mol. Microbiol. 1999; 34: 867-877Crossref PubMed Scopus (269) Google Scholar). Helicases are generally believed to generate single-stranded DNA by catalyzing the melting of stable helical DNA structures utilizing energy derived from the hydrolysis of nucleoside triphosphates. Analysis of the genome of Saccharomyces cerevisiae indicates that there are 134 open reading frames containing with helicase-like features that represent ∼2% of its genome (7Shiratori A. Shibata T. Arisawa M. Hanaoka F. Murakami Y. Eki T. Yeast. 1999; 15: 219-253Crossref PubMed Scopus (93) Google Scholar). The presence of such a large number of helicases or helicase-like proteins in cells, many of unknown function in vivo, most likely reflects a complexity of nucleic acid metabolic reactions and the distinct structural template requirements for a given helicase. Alternatively, many helicases may play roles that functionally overlap, making it difficult to determine a specific role for a given helicase. For example, each mutant cell of the two yeast helicases, Sgs1 and Srs2, grows with wild-type kinetics, but the combination of an sgs1 mutation with loss of the helicase srs2 resulted in a severe growth defect (8Lee S.K. Johnson R.E., Yu, S.L. Prakash L. Prakash S. Science. 1999; 286: 2339-2342Crossref PubMed Scopus (126) Google Scholar, 9McVey M. Kaeberlein M. Tissenbaum H.A. Guarente L. Genetics. 2001; 157: 1531-1542Crossref PubMed Google Scholar). A majority of sgs1 and srs2 mutant cells stops dividing stochastically as large budded DNA (9McVey M. Kaeberlein M. Tissenbaum H.A. Guarente L. Genetics. 2001; 157: 1531-1542Crossref PubMed Google Scholar). Such double mutant cells are unable to replicate DNA at restrictive temperature and unable to efficiently transcribe rDNA (8Lee S.K. Johnson R.E., Yu, S.L. Prakash L. Prakash S. Science. 1999; 286: 2339-2342Crossref PubMed Scopus (126) Google Scholar). This suggests that these two helicases provide a redundant but essential activity for DNA replication and rDNA transcription. Despite well conserved motifs specific to helicase family proteins, there are several proteins for which no helicase activity has been demonstrated, indicating that helicase motifs cannot, without additional information, be used to define a protein as a helicase. For example, the chromatin-remodeling factor SWI2/SNF2 containing these motifs lacks helicase activity, although its ability to hydrolyze ATP is stimulated by DNA (10Richmond E. Peterson C.L. Nucleic Acids Res. 1996; 24: 3685-3692Crossref PubMed Scopus (110) Google Scholar, 11Travers A. Cell. 1999; 96: 311-314Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), suggesting that ATP hydrolysis provides the energy required to alter protein-DNA structure rather than duplex DNA or RNA structure. This suggests that proteins with helicase motifs may have functions that do not involve unwinding of nucleic acids. As a continued effort to understand the role ofSchizosaccharomyces pombe DNA helicase I that we reported previously (12Park J.S. Choi E. Lee S.H. Lee C. Seo Y.S. J. Biol. Chem. 1997; 272: 18910-18919Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), we identified a human homolog of this fission yeast enzyme and named it hFBH1 (humanF-box DNA helicase 1) because it contains a well conserved functional F-box motif (13Bai C. Sen P. Hofmann K., Ma, L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). In this report, we present data that hFBH1 is not only an ATPase/DNA helicase but also is an F-box protein that can form an SCF (SKP1-CUL1 (Cdc53)-Rbx1-F-box protein) complex with human SKP1 and CUL1. SCF complexes constitute a new class of E31 ligase that plays important roles in cell cycle regulation and signal transduction by catalyzing ubiquitin-mediated proteolysis (14Peters J.M. Curr. Opin. Cell Biol. 1998; 10: 759-768Crossref PubMed Scopus (224) Google Scholar, 15Craig K.L. Tyers M. Prog. Biophys. Mol. Biol. 1999; 72: 299-328Crossref PubMed Scopus (236) Google Scholar, 16Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 21: 1180-1182Abstract Full Text Full Text PDF Scopus (305) Google Scholar, 17Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 18Tyers M. Jorgensen P. Curr. Opin. Genet. Dev. 2000; 10: 54-64Crossref PubMed Scopus (268) Google Scholar). The substrate specificity of an SCF complex is governed by the interchangeable F-box protein subunit, which recruits a specific set of substrates for ubiquitination to the SCF core complex composed of SKP1, Cdc53, ROC1, and the E2 enzyme Cdc34. The polyubiquitinated proteins are rapidly captured by the 26 S proteasome, an abundant, self-compartmentalized protease particle (19Baumeister W. Walz J. Zuhl F. Seemuller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). To date, hFBH1 is the first example of an F-box protein that contains intrinsic enzymatic activity, suggesting that a helicase can play a role in a certain aspect of DNA metabolism that requires ubiquitin-dependent proteolysis. The potential biological role of this interesting human DNA helicase will be discussed. DISCUSSIONIn this report, we demonstrate that hFBH1 is a DNA helicase that translocates in the 3′ to 5′ direction and interacts with SKP1 in a manner that requires a functional F-box motif. Besides, hFBH1 interacted with CUL1/ROC1 and formed a SCF complex that contained all known four subunits (SKP1, hFBH1, CUL1, and ROC1). Moreover, SCFhFBH1 displayed ubiquitin ligase activity in a F-box motif-dependent fashion. The polyubiquitination activity of the SCFhFBH1 complex was dependent upon functional E1 and E2 enzymes, demonstrating that the complex interacts with the ubiquitination machinery. Our study showed that hFBH1 is the first example of an F-box protein that harbors an intrinsic enzymatic activity, catalyzing a helicase reaction. In addition, hFBH1 is the first F-box protein implicated in nucleic acid metabolism. Therefore, our findings raise the possibility that the role of hFBH1 is most likely to recruit a target protein, most likely involved in some aspect of DNA metabolism, to the protein degradation pathway. Thus, a protein involved in DNA metabolism would be marked by hFBH1 for degradation via the highly regulated ubiquitin degradation pathway.As an initial attempt to evaluate the role of the F-box motif of the enzyme in this regard, we decided to identify a protein(s) that interacts specifically with hFBH1 using the yeast two-hybrid screen. This screen resulted in the isolation of human MAT1 (data not shown), which is a regulatory subunit of the Cdk-activating kinase (29Yee A. Nichols M.A., Wu, L. Hall F.L. Kobayashi R. Xiong Y. Cancer Res. 1995; 55: 6058-6062PubMed Google Scholar, 30Yee A., Wu, L. Liu L. Kobayashi R. Xiong Y. Hall F.L. J. Biol. Chem. 1996; 271: 471-477Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). It is unlikely that MAT1 is a direct substrate of SCFhFBH1 E3 ligase activity because a substrate phosphorylation is a prerequisite step prior to recognition by an F-box protein (31Elsasser S. Chi Y. Yang P. Campbell J.L. Mol. Biol. Cell. 1999; 10: 3263-3277Crossref PubMed Scopus (134) Google Scholar, 32Deshaies R.J. Curr. Opin. Genet. Dev. 1997; 7: 7-16Crossref PubMed Scopus (82) Google Scholar). MAT1 in Cdk-activating kinase may play a role leading to the phosphorylation of a substrate in proximity with hFBH1, thereby facilitating the subsequent ubiquitination of the substrate by the SCFhFBH1complex. In support of this possibility, we failed to detect any ubiquitination of MAT1 (data not shown). These findings raise the possibility that MAT1 may play a direct role in the phosphorylation of hFBH1 instead of acting as a substrate for the E3 ligase activity of the SCFhFBH1 complex. Therefore, the level of hFBH1 itself is likely to be regulated by posttranslational modifications that involve both Cdk-activating kinase and E3 ligase activities. Because it has been reported that F-box proteins can be degraded autocatalytically in a ubiquitination-dependent fashion (33Galan J.M. Peter M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9124-9129Crossref PubMed Scopus (226) Google Scholar), the F-box motif of hFBH1 may regulate the level of hFBH1 through auto-ubiquitination during cell cycle progression. However, we were not able to demonstrate this, because the endogenous levels of hFBH1 were too low to be detected with the antibodies that we have (data not shown). We also explored whether hFBH1 is ubiquitinated in vivo. Although the hFBH1 was indeed ubiquitinated in vivo (data not shown), the efficiency of hFBH1 ubiquitination was independent of the intact F-box motif, suggesting that ubiquitination occurred in trans by some other ubiquitinating activity. Reconstitution of the SCF complex containing hFBH1 with the purified proteins will help to address this question.Although the biological function in vivo of hFBH1 is unclear at present, mutational studies of the S. pombe fdh1 + gene (encoding the S. pombe homolog of hFBH1) have provided some clues. When the S. pombe fdh1 + gene was deleted, the cells showed elongated cell morphology and uneven distribution of chromosomes in dividing cells. 2S. H. Lee and Y. S. Seo, unpublished observation. In addition, deletion of either the F-box motif or a single amino acid change in the ATP-binding motif resulted in phenotypes similar to those of the null mutation.2 These observations suggest that both the F-box and ATPase/helicase activity of hFBH1 are required for the physiological function of the enzyme. Currently, both genetic and biochemical studies are underway using an S. pombe model system to delineate the precise biological role of hFBH1. In this report, we demonstrate that hFBH1 is a DNA helicase that translocates in the 3′ to 5′ direction and interacts with SKP1 in a manner that requires a functional F-box motif. Besides, hFBH1 interacted with CUL1/ROC1 and formed a SCF complex that contained all known four subunits (SKP1, hFBH1, CUL1, and ROC1). Moreover, SCFhFBH1 displayed ubiquitin ligase activity in a F-box motif-dependent fashion. The polyubiquitination activity of the SCFhFBH1 complex was dependent upon functional E1 and E2 enzymes, demonstrating that the complex interacts with the ubiquitination machinery. Our study showed that hFBH1 is the first example of an F-box protein that harbors an intrinsic enzymatic activity, catalyzing a helicase reaction. In addition, hFBH1 is the first F-box protein implicated in nucleic acid metabolism. Therefore, our findings raise the possibility that the role of hFBH1 is most likely to recruit a target protein, most likely involved in some aspect of DNA metabolism, to the protein degradation pathway. Thus, a protein involved in DNA metabolism would be marked by hFBH1 for degradation via the highly regulated ubiquitin degradation pathway. As an initial attempt to evaluate the role of the F-box motif of the enzyme in this regard, we decided to identify a protein(s) that interacts specifically with hFBH1 using the yeast two-hybrid screen. This screen resulted in the isolation of human MAT1 (data not shown), which is a regulatory subunit of the Cdk-activating kinase (29Yee A. Nichols M.A., Wu, L. Hall F.L. Kobayashi R. Xiong Y. Cancer Res. 1995; 55: 6058-6062PubMed Google Scholar, 30Yee A., Wu, L. Liu L. Kobayashi R. Xiong Y. Hall F.L. J. Biol. Chem. 1996; 271: 471-477Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). It is unlikely that MAT1 is a direct substrate of SCFhFBH1 E3 ligase activity because a substrate phosphorylation is a prerequisite step prior to recognition by an F-box protein (31Elsasser S. Chi Y. Yang P. Campbell J.L. Mol. Biol. Cell. 1999; 10: 3263-3277Crossref PubMed Scopus (134) Google Scholar, 32Deshaies R.J. Curr. Opin. Genet. Dev. 1997; 7: 7-16Crossref PubMed Scopus (82) Google Scholar). MAT1 in Cdk-activating kinase may play a role leading to the phosphorylation of a substrate in proximity with hFBH1, thereby facilitating the subsequent ubiquitination of the substrate by the SCFhFBH1complex. In support of this possibility, we failed to detect any ubiquitination of MAT1 (data not shown). These findings raise the possibility that MAT1 may play a direct role in the phosphorylation of hFBH1 instead of acting as a substrate for the E3 ligase activity of the SCFhFBH1 complex. Therefore, the level of hFBH1 itself is likely to be regulated by posttranslational modifications that involve both Cdk-activating kinase and E3 ligase activities. Because it has been reported that F-box proteins can be degraded autocatalytically in a ubiquitination-dependent fashion (33Galan J.M. Peter M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9124-9129Crossref PubMed Scopus (226) Google Scholar), the F-box motif of hFBH1 may regulate the level of hFBH1 through auto-ubiquitination during cell cycle progression. However, we were not able to demonstrate this, because the endogenous levels of hFBH1 were too low to be detected with the antibodies that we have (data not shown). We also explored whether hFBH1 is ubiquitinated in vivo. Although the hFBH1 was indeed ubiquitinated in vivo (data not shown), the efficiency of hFBH1 ubiquitination was independent of the intact F-box motif, suggesting that ubiquitination occurred in trans by some other ubiquitinating activity. Reconstitution of the SCF complex containing hFBH1 with the purified proteins will help to address this question. Although the biological function in vivo of hFBH1 is unclear at present, mutational studies of the S. pombe fdh1 + gene (encoding the S. pombe homolog of hFBH1) have provided some clues. When the S. pombe fdh1 + gene was deleted, the cells showed elongated cell morphology and uneven distribution of chromosomes in dividing cells. 2S. H. Lee and Y. S. Seo, unpublished observation. In addition, deletion of either the F-box motif or a single amino acid change in the ATP-binding motif resulted in phenotypes similar to those of the null mutation.2 These observations suggest that both the F-box and ATPase/helicase activity of hFBH1 are required for the physiological function of the enzyme. Currently, both genetic and biochemical studies are underway using an S. pombe model system to delineate the precise biological role of hFBH1. We thank Dr. J. Hurwitz (Sloan-Kettering Institute, New York, NY) for critical reading of the manuscript." @default.
• W1967110156 created "2016-06-24" @default.
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• W1967110156 date "2002-07-01" @default.
• W1967110156 modified "2023-10-01" @default.
• W1967110156 title "The Novel Human DNA Helicase hFBH1 Is an F-box Protein" @default.
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