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Characterization of the Net1 Cell Cycle-dependent Regulator of the Cdc14 Phosphatase from Budding Yeast*

  • Edwin E. Traverso
    Footnotes
    Affiliations
    From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, the
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  • Christopher Baskerville
    Footnotes
    Affiliations
    From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, the
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  • Yan Liu
    Affiliations
    From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, the
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  • Wenying Shou
    Affiliations
    Division of Biology, California Institute of Technology, Pasadena, California 91125, and the
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  • Philip James
    Footnotes
    Affiliations
    Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, 53706
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  • Raymond J. Deshaies
    Affiliations
    Division of Biology, California Institute of Technology, Pasadena, California 91125, and the
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  • Harry Charbonneau
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, the
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant CA59935 (to H. C.), a Howard Hughes Medical Institute predoctoral fellowship (to W. S.), a Beckman Young Investigator award (to R. J. D), and a fellowship (to Y. L.) from the Indiana Elks Charities. This is Journal Paper 16424 from the Purdue University Agriculture Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Both authors contributed equally to this work.
    ** Work in the laboratory of Dr. Elizabeth A. Craig was supported by National Institutes of Health Grant GM31107.
Open AccessPublished:June 15, 2001DOI:https://doi.org/10.1074/jbc.M011689200
      In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase. Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed. Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm. In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We show that the NH2-terminal fragment Net1(1–600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (K i = 3 nm) with five different substrates including the physiologic targets Swi5 and Sic1. An analysis of truncation mutants indicates that the Cdc14 binding site is located within a segment of Net1 containing residues 1–341. We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1–374), binds with reduced affinity to a Cdc14 C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein-tyrosine phosphatases.
      PTP
      protein-tyrosine phosphatase
      APC/C
      anaphase-promoting complex/cyclosome
      SC
      synthetic complete
      GST
      glutathioneS-transferase
      PEI
      polyethyleneimine
      HCdc14
      human homologs of yeast Cdc14
      HA
      hemagglutinin
      PAGE
      polyacrylamide gel electrophoresis
      MBP
      myelin basic protein
      pNPP
      p-nitrophenyl phosphate
      Tyr(P)-MBP
      myelin basic protein phosphorylated on tyrosine residues
      The Cdc14 phosphatases are a conserved subset of dual specificity enzymes (
      • Taylor G.S.
      • Liu Y.
      • Baskerville C.
      • Charbonneau H.
      ,
      • Li L.
      • Ernsting B.R.
      • Wishart M.J.
      • Lohse D.L.
      • Dixon J.E.
      ,
      • Li L.
      • Ljungman M.
      • Dixon J.E.
      ) of the protein-tyrosine phosphatase (PTP)1 family (
      • Barford D.
      • Das A.K.
      • Egloff M.P.
      ,
      • Neel B.G.
      • Tonks N.K.
      ,
      • Fauman E.B.
      • Saper M.A.
      ,
      • Tonks N.K.
      • Neel B.G.
      ). The essential Cdc14 phosphatase from budding yeast (
      • Taylor G.S.
      • Liu Y.
      • Baskerville C.
      • Charbonneau H.
      ,
      • Visintin R.
      • Craig K.
      • Hwang E.S.
      • Prinz S.
      • Tyers M.
      • Amon A.
      ,
      • Jaspersen S.L.
      • Charles J.F.
      • Tinker-Kulberg R.L.
      • Morgan D.O.
      ,
      • Jaspersen S.L.
      • Charles J.F.
      • Morgan D.O.
      ),Saccharomyces cerevisiae, is involved in driving cells from late anaphase into G1 of the subsequent cell cycle, a series of events known as exit from mitosis (for review, see Refs.
      • Cerutti L.
      • Simanis V.
      ,
      • Morgan D.O.
      ,
      • Zachariae W.
      ). The onset of mitosis occurs when cyclin-dependent kinases are activated following their association with mitotic cyclins. Exit from mitosis requires the inactivation of cyclin-dependent kinases, a process that involves the ubiquitination and subsequent destruction of cyclins and other regulatory proteins. The anaphase-promoting complex/cyclosome (APC/C) is a tightly regulated multisubunit ubiquitin ligase that first initiates anaphase, then exit from mitosis, by targeting proteins for degradation in an ordered and tightly coordinated fashion (for review, see Ref.
      • Zachariae W.
      • Nasmyth K.
      ). In budding yeast, inactivation of the mitotic cyclin-dependent kinase (Cdc28) occurs by two processes, the APC/C-dependent ubiquitination and subsequent destruction of B-type cyclins (Clb1–6) and the synthesis of the Clb/Cdc28 inhibitor Sic1 (
      • Morgan D.O.
      ,
      • Zachariae W.
      ). Cdc14 drives both of these processes by dephosphorylating at least three targets: Hct1, Swi5, and Sic1 (
      • Visintin R.
      • Craig K.
      • Hwang E.S.
      • Prinz S.
      • Tyers M.
      • Amon A.
      ,
      • Jaspersen S.L.
      • Charles J.F.
      • Morgan D.O.
      ). Cdc14 dephosphorylates inhibitory sites and thereby activates the APC/C regulator Cdh1/Hct1 so that a subset of mitotic cyclins, Clb2 and Clb3, is targeted for ubiquitination by the APC/C (
      • Visintin R.
      • Craig K.
      • Hwang E.S.
      • Prinz S.
      • Tyers M.
      • Amon A.
      ,
      • Jaspersen S.L.
      • Charles J.F.
      • Morgan D.O.
      ). Swi5 is a zinc finger transcription factor that is required for expression of theSIC1 gene. The dephosphorylation of Swi5 permits its translocation from the cytoplasm to the nucleus where it can activate Sic1 transcription (
      • Visintin R.
      • Craig K.
      • Hwang E.S.
      • Prinz S.
      • Tyers M.
      • Amon A.
      ). The Sic1 protein itself may be protected from premature degradation when dephosphorylated by Cdc14 (
      • Visintin R.
      • Craig K.
      • Hwang E.S.
      • Prinz S.
      • Tyers M.
      • Amon A.
      ).
      Net1 (also known as Cfi1) is a core component of the nucleolar RENT complex that regulates Cdc14 during the cell cycle (for review, see Refs.
      • Visintin R.
      • Amon A.
      ,
      • Cockell M.M.
      • Gasser S.M.
      ,
      • Garcia S.N.
      • Pillus L.
      ). From G1 to anaphase, Net1 sequesters Cdc14 in the nucleolus, where its access to substrate is limited, and its phosphatase activity is suppressed (
      • Shou W.
      • Seol J.H.
      • Shevchenko A.
      • Baskerville C.
      • Moazed D.
      • Chen Z.W.
      • Jang J.
      • Charbonneau H.
      • Deshaies R.J.
      ,
      • Visintin R.
      • Hwang E.S.
      • Amon A.
      ,
      • de Almeida A.
      • Raccurt I.
      • Peyrol S.
      • Charbonneau M.
      ). At late anaphase, Cdc14 is transiently released from Net1 permitting the active phosphatase to reach targets in the nucleus and cytoplasm (
      • Shou W.
      • Seol J.H.
      • Shevchenko A.
      • Baskerville C.
      • Moazed D.
      • Chen Z.W.
      • Jang J.
      • Charbonneau H.
      • Deshaies R.J.
      ,
      • Visintin R.
      • Hwang E.S.
      • Amon A.
      ). The RENT complex appears to have multiple functions besides Cdc14 regulation including roles in maintaining the integrity of the nucleolus (
      • de Almeida A.
      • Raccurt I.
      • Peyrol S.
      • Charbonneau M.
      ,
      • Straight A.F.
      • Shou W.
      • Dowd G.J.
      • Turck C.W.
      • Deshaies R.J.
      • Johnson A.D.
      • Moazed D.
      ) and sequestering Sir2 to tandem rDNA repeats (
      • Straight A.F.
      • Shou W.
      • Dowd G.J.
      • Turck C.W.
      • Deshaies R.J.
      • Johnson A.D.
      • Moazed D.
      ). The NAD-dependent histone deacetylase activity of Sir2 silences rDNA chromatin and represses recombination among tandem rDNA repeats, a deleterious process that leads to senescence of cells (
      • Guarente L.
      ,
      • Gartenberg M.R.
      ,
      • Gottschling D.E.
      ).
      The mechanism by which Cdc14 is released from Net1 is not yet clear, but it is dependent in part on activation of a signaling pathway known as the mitotic exit network (
      • Jaspersen S.L.
      • Charles J.F.
      • Tinker-Kulberg R.L.
      • Morgan D.O.
      ,
      • Cerutti L.
      • Simanis V.
      ,
      • Morgan D.O.
      ). When the dividing nucleus spans the bud neck during late mitosis, the mitotic exit network pathway is activated, and a signal is propagated which promotes release of Cdc14 (
      • Bardin A.J.
      • Visintin R.
      • Amon A.
      ,
      • Pereira G.
      • Hofken T.
      • Grindlay J.
      • Manson C.
      • Schiebel E.
      ). The APC/C-mediated destruction of the anaphase inhibitor Pds1 is not only necessary for sister-chromatid separation but also for the subsequent release of Cdc14 (
      • Cohen-Fix O.
      • Koshland D.
      ,
      • Shirayama M.
      • Toth A.
      • Galova M.
      • Nasmyth K.
      ,
      • Tinker-Kulberg R.L.
      • Morgan D.O.
      ). However, Pds1 degradation and Cdc14 release are not sufficient for exit from mitosis unless the Clb5 cyclin has also been destroyed by the APC/C at the metaphase to anaphase transition (
      • Shirayama M.
      • Toth A.
      • Galova M.
      • Nasmyth K.
      ). Thus, multiple controls ensure that exit from mitosis occurs only after chromosome segregation and correct partitioning of the dividing nucleus to the mother and daughter cells (
      • Bardin A.J.
      • Visintin R.
      • Amon A.
      ,
      • Pereira G.
      • Hofken T.
      • Grindlay J.
      • Manson C.
      • Schiebel E.
      ,
      • Shirayama M.
      • Toth A.
      • Galova M.
      • Nasmyth K.
      ,
      • Tinker-Kulberg R.L.
      • Morgan D.O.
      ,
      • Wang Y.
      • Hu F.
      • Elledge S.J.
      ).
      Previous studies have not determined whether Net1 on its own is sufficient to inhibit Cdc14 activity. In this paper we have characterized the direct effect of Net1 on Cdc14 activity in vitro. We have shown that a 600-residue NH2-terminal fragment of Net1 alone binds the catalytic domain of Cdc14 and acts as a potent and specific competitive inhibitor of its activity toward physiologic substrates. We have defined a segment of Net1 spanning residues 1–341 which contains the Cdc14 binding site and fully inhibits phosphatase activity. We also provide evidence that Net1 acts by occluding the active site of Cdc14.

      DISCUSSION

      Current models (
      • Visintin R.
      • Amon A.
      ,
      • Cockell M.M.
      • Gasser S.M.
      ,
      • Garcia S.N.
      • Pillus L.
      ,
      • Shou W.
      • Seol J.H.
      • Shevchenko A.
      • Baskerville C.
      • Moazed D.
      • Chen Z.W.
      • Jang J.
      • Charbonneau H.
      • Deshaies R.J.
      ) posit that Net1 retains inactive Cdc14 in the nucleolus during interphase and early mitosis until the transient release of the active phosphatase at late anaphase triggers exit from mitosis. The intrinsic biochemical properties of Net1(1–600) which we have observed in vitro fully support this model. Nanomolar concentrations of Net1(1–600) alone fully inhibited the activity of Cdc14 through a direct interaction that was not dependent on other proteins. The effect of Net1(1–600) was specific to Cdc14 and observed with all substrates examined including the physiologic targets, Swi5 and Sic1. Importantly, Net1(1–600) retained its ability to act as a high affinity inhibitor (IC50 = 70 nm) when buffers approximating physiologic ionic strength and pH were employed. These findings demonstrate that Net1 alone has the capacity to regulate all cellular functions requiring Cdc14 by sequestering the phosphatase from the nucleoplasm to the nucleolus and fully inhibiting its activity. Our results also suggest that post-translational modifications of the type commonly observed in eukaryotes are unlikely to be required for inhibition because Net1(1–600) was produced in bacteria. However, post-translational modification or the binding of other protein ligands could influence the affinity of Net1 for Cdc14 and may mediate the release of Cdc14 at late anaphase as suggested previously (
      • Shou W.
      • Seol J.H.
      • Shevchenko A.
      • Baskerville C.
      • Moazed D.
      • Chen Z.W.
      • Jang J.
      • Charbonneau H.
      • Deshaies R.J.
      ).
      Results from in vitro binding, inhibition assays, and limited proteolytic cleavage demonstrate that residues 1–341 of Net1 are sufficient for both binding and inhibiting Cdc14 (Fig.5 C). Two-hybrid assays suggest that the first 91 residues of Net1 are not essential for binding, but the possibility that this segment is needed for inhibition has not been excluded. Net1 also sequesters Sir2 within the nucleolus where it is involved in rDNA silencing. Cuperus et al. (
      • Cuperus G.
      • Shafaatian R.
      • Shore D.
      ) have shown that residues 566–801 of Net1 interact with an NH2-terminal fragment of Sir2 (Fig. 5 C). Their results together with our data establish that Sir2 and Cdc14 bind to distinct, non-overlapping sites within the linear sequence of Net1 and show that neither protein is required for binding of the other (Fig. 5 C). Additional structure-function analyses of Net1 will be required to define the minimal sequence(s) required for binding and inhibiting Cdc14.
      Several findings suggest that Net1 inhibits by occluding the active site of Cdc14. Net1 acts as a competitive inhibitor of Cdc14, indicating that inhibitor and substrate binding are mutually exclusive. The Net1 binding site has been mapped to a position within the conserved domain of Cdc14 (residues 1–374) which encompasses the entire active site region (residues 279–291). The structures (
      • Barford D.
      • Das A.K.
      • Egloff M.P.
      ,
      • Barford D.
      • Flint A.J.
      • Tonks N.K.
      ,
      • Fauman E.B.
      • Yuvaniyama C.
      • Schubert H.L.
      • Stuckey J.A.
      • Saper M.A.
      ,
      • Stuckey J.A.
      • Schubert H.L.
      • Fauman E.B.
      • Zhang Z.Y.
      • Dixon J.E.
      • Saper M.A.
      ,
      • Yang J.
      • Liang X.
      • Niu T.
      • Meng W.
      • Zhao Z.
      • Zhou G.W.
      ) of several different PTPs reveal that tungstate, a general PTP inhibitor that mimics the transition state, is bound within the active site in a manner similar to the phosphate of substrates. Sodium tungstate prevents Net1 binding in a concentration-dependent manner (Fig. 3), suggesting that contacts within the Cdc14 active site are required for binding. The significant reduction in Net1 affinity which occurs upon replacement of the Cdc14 active site cysteine also supports the idea that binding involves the catalytic site. Several studies indicate that replacement of the active site Cys can induce local changes in the conformation and properties of PTP catalytic sites (
      • Zhang Y.L.
      • Yao Z.J.
      • Sarmiento M.
      • Wu L.
      • Burke Jr., T.R.
      • Zhang Z.Y.
      ,
      • Zhang Z.Y.
      • Wu L.
      ). For example, aYersinia PTP mutant, in which the analogous Cys is replaced by Ser, exhibits a 10-fold reduction in affinity for tungstate (
      • Zhang Z.Y.
      • Wu L.
      ). Although we propose an interaction within the catalytic cleft of Cdc14, it is likely that contacts with flanking surfaces are also involved. Interactions with adjacent surfaces could account for the selectivity of Net1 for the yeast enzyme over human Cdc14 and other PTPs despite the high degree of sequence similarity in their catalytic sites. These data favor a model in which Net1 binds at the active site, but we cannot rule out the possibility that it binds to a distinct inhibitory site and competes indirectly through long range interactions that result in conformational changes at the substrate binding site. Additional studies will be required to delineate further the precise location of the Net1 binding site.
      Residues 1–220 of Net1 exhibit 30% sequence identity to residues 1–233 of another yeast protein Tof2 (Fig. 5 C). Other than its ability to interact with DNA topoisomerase I (
      • Park H.
      • Sternglanz R.
      ), little is known about the function of Tof2. Although the Tof2-like segment is contained within the Cdc14 binding region defined by these studies (Fig. 5 C), it is not yet clear whether these sequences are involved directly in binding and/or inhibition. The lack of binding observed with the 27-kDa cleavage product of Net1, which spans most if not all of these conserved sequences, suggests the Tof2-like region may not have a direct role in regulating Cdc14.
      The Caenorhabditis elegans, Drosophila, and human genomes and the sequence data bases contain no open reading frames exhibiting significant sequence similarity to Net1. The lack of an extant Net1 homolog in metazoans is surprising because Cdc14 phosphatase genes are found in the C. elegans,Drosophila, zebrafish, chicken, mouse, and human genomes. This observation coupled with the insensitivity of human Cdc14 to Net1 suggests that the regulation of metazoan and budding yeast Cdc14 differ. In contrast to fungi, the nuclear membrane and nucleolus of metazoans break down during mitosis. As a result of its disassembly, higher eukaryotes may not employ the nucleolus as a site to regulate exit from mitosis. It will be important to determine how Cdc14 activity and its potential function in mitotic exit are controlled in metazoans.

      Acknowledgments

      We thank Drs. S. Rossie and Z.-Y. Zhang for generously providing samples of the PPT1 and VHR protein phosphatases, respectively; the Purdue Laboratory for Macromolecular Structure for performing amino acid sequence analyses; and Dr. S. Rossie for critical comments on the manuscript.

      REFERENCES

        • Taylor G.S.
        • Liu Y.
        • Baskerville C.
        • Charbonneau H.
        J. Biol. Chem. 1997; 272: 24054-24063
        • Li L.
        • Ernsting B.R.
        • Wishart M.J.
        • Lohse D.L.
        • Dixon J.E.
        J. Biol. Chem. 1997; 272: 29403-29406
        • Li L.
        • Ljungman M.
        • Dixon J.E.
        J. Biol. Chem. 2000; 275: 2410-2414
        • Barford D.
        • Das A.K.
        • Egloff M.P.
        Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 133-164
        • Neel B.G.
        • Tonks N.K.
        Curr. Opin. Cell Biol. 1997; 9: 193-204
        • Fauman E.B.
        • Saper M.A.
        Trends Biochem. Sci. 1996; 21: 413-417
        • Tonks N.K.
        • Neel B.G.
        Cell. 1996; 87: 365-368
        • Visintin R.
        • Craig K.
        • Hwang E.S.
        • Prinz S.
        • Tyers M.
        • Amon A.
        Mol. Cell. 1998; 2: 709-718
        • Jaspersen S.L.
        • Charles J.F.
        • Tinker-Kulberg R.L.
        • Morgan D.O.
        Mol. Biol. Cell. 1998; 9: 2803-2817
        • Jaspersen S.L.
        • Charles J.F.
        • Morgan D.O.
        Curr. Biol. 1999; 9: 227-236
        • Cerutti L.
        • Simanis V.
        Curr. Opin. Genet. Dev. 2000; 10: 65-69
        • Morgan D.O.
        Nat. Cell Biol. 1999; 1: E47-E53
        • Zachariae W.
        Curr. Opin. Cell Biol. 1999; 11: 708-716
        • Zachariae W.
        • Nasmyth K.
        Genes Dev. 1999; 13: 2039-2058
        • Visintin R.
        • Amon A.
        Curr. Opin. Cell Biol. 2000; 12: 372-377
        • Cockell M.M.
        • Gasser S.M.
        Curr. Biol. 1999; 9: R575-R576
        • Garcia S.N.
        • Pillus L.
        Cell. 1999; 97: 825-828
        • Shou W.
        • Seol J.H.
        • Shevchenko A.
        • Baskerville C.
        • Moazed D.
        • Chen Z.W.
        • Jang J.
        • Charbonneau H.
        • Deshaies R.J.
        Cell. 1999; 97: 233-244
        • Visintin R.
        • Hwang E.S.
        • Amon A.
        Nature. 1999; 398: 818-823
        • de Almeida A.
        • Raccurt I.
        • Peyrol S.
        • Charbonneau M.
        Biol. Cell. 1999; 91: 649-663
        • Straight A.F.
        • Shou W.
        • Dowd G.J.
        • Turck C.W.
        • Deshaies R.J.
        • Johnson A.D.
        • Moazed D.
        Cell. 1999; 97: 245-256
        • Guarente L.
        Genes Dev. 2000; 14: 1021-1026
        • Gartenberg M.R.
        Curr. Opin. Microbiol. 2000; 3: 132-137
        • Gottschling D.E.
        Curr. Biol. 2000; 10: R708-R711
        • Bardin A.J.
        • Visintin R.
        • Amon A.
        Cell. 2000; 102: 21-31
        • Pereira G.
        • Hofken T.
        • Grindlay J.
        • Manson C.
        • Schiebel E.
        Mol. Cell. 2000; 6: 1-10
        • Cohen-Fix O.
        • Koshland D.
        Genes Dev. 1999; 13: 1950-1959
        • Shirayama M.
        • Toth A.
        • Galova M.
        • Nasmyth K.
        Nature. 1999; 402: 203-207
        • Tinker-Kulberg R.L.
        • Morgan D.O.
        Genes Dev. 1999; 13: 1936-1949
        • Wang Y.
        • Hu F.
        • Elledge S.J.
        Curr. Biol. 2000; 10: 1379-1382
        • James P.
        • Halladay J.
        • Craig E.A.
        Genetics. 1996; 144: 1425-1436
        • Hao L.
        • Tiganis T.
        • Tonks N.K.
        • Charbonneau H.
        J. Biol. Chem. 1997; 272: 29322-29329
        • Verma R.
        • Annan R.S.
        • Huddleston M.J.
        • Carr S.A.
        • Reynard G.
        • Deshaies R.J.
        Science. 1997; 278: 455-460
        • Futcher B.
        Fantes P. Brooks R. The Cell Cycle: A Practical Approach. Oxford University Press, New York1993: 80-84
        • Sheng Z.
        • Charbonneau H.
        J. Biol. Chem. 1993; 268: 4728-4733
        • Matsudaira P.
        J. Biol. Chem. 1987; 262: 10035-10038
        • Henderson P.J.
        Biochem. J. 1972; 127: 321-333
        • Moll T.
        • Tebb G.
        • Surana U.
        • Robitsch H.
        • Nasmyth K.
        Cell. 1991; 66: 743-758
        • Zhang Z.Y.
        • Wu L.
        • Chen L.
        Biochemistry. 1995; 34: 16088-16096
        • Chen M.X.
        • McPartlin A.E.
        • Brown L.
        • Chen Y.H.
        • Barker H.M.
        • Cohen P.T.
        EMBO J. 1994; 13: 4278-4290
        • Cuperus G.
        • Shafaatian R.
        • Shore D.
        EMBO J. 2000; 19: 2641-2651
        • Barford D.
        • Flint A.J.
        • Tonks N.K.
        Science. 1994; 263: 1397-1404
        • Fauman E.B.
        • Yuvaniyama C.
        • Schubert H.L.
        • Stuckey J.A.
        • Saper M.A.
        J. Biol. Chem. 1996; 271: 18780-18788
        • Stuckey J.A.
        • Schubert H.L.
        • Fauman E.B.
        • Zhang Z.Y.
        • Dixon J.E.
        • Saper M.A.
        Nature. 1994; 370: 571-575
        • Yang J.
        • Liang X.
        • Niu T.
        • Meng W.
        • Zhao Z.
        • Zhou G.W.
        J. Biol. Chem. 1998; 273: 28199-28207
        • Zhang Y.L.
        • Yao Z.J.
        • Sarmiento M.
        • Wu L.
        • Burke Jr., T.R.
        • Zhang Z.Y.
        J. Biol. Chem. 2000; 275: 34205-34212
        • Zhang Z.Y.
        • Wu L.
        Biochemistry. 1997; 36: 1362-1369
        • Park H.
        • Sternglanz R.
        Yeast. 1999; 15: 35-41
        • Bradford M.M.
        Anal. Biochem. 1976; 72: 248-254