Characterization of the Net1 Cell Cycle-dependent Regulator of the Cdc14 Phosphatase from Budding Yeast
2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês
10.1074/jbc.m011689200
ISSN1083-351X
AutoresEdwin E. Traverso, Christopher Baskerville, Yan Liu, Wenying Shou, Philip James, Raymond J. Deshaies, Harry Charbonneau,
Tópico(s)Fungal and yeast genetics research
ResumoIn 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. 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. protein-tyrosine phosphatase anaphase-promoting complex/cyclosome synthetic complete glutathioneS-transferase polyethyleneimine human homologs of yeast Cdc14 hemagglutinin polyacrylamide gel electrophoresis myelin basic protein p-nitrophenyl phosphate myelin basic protein phosphorylated on tyrosine residues The Cdc14 phosphatases are a conserved subset of dual specificity enzymes (1Taylor G.S. Liu Y. Baskerville C. Charbonneau H. J. Biol. Chem. 1997; 272: 24054-24063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 2Li L. Ernsting B.R. Wishart M.J. Lohse D.L. Dixon J.E. J. 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Prinz S. Tyers M. Amon A. Mol. Cell. 1998; 2: 709-718Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 9Jaspersen S.L. Charles J.F. Tinker-Kulberg R.L. Morgan D.O. Mol. Biol. Cell. 1998; 9: 2803-2817Crossref PubMed Scopus (263) Google Scholar, 10Jaspersen S.L. Charles J.F. Morgan D.O. Curr. Biol. 1999; 9: 227-236Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar),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.11Cerutti L. Simanis V. Curr. Opin. Genet. Dev. 2000; 10: 65-69Crossref PubMed Scopus (42) Google Scholar, 12Morgan D.O. Nat. Cell Biol. 1999; 1: E47-E53Crossref PubMed Scopus (306) Google Scholar, 13Zachariae W. Curr. Opin. Cell Biol. 1999; 11: 708-716Crossref PubMed Scopus (41) Google Scholar). 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. 14Zachariae W. Nasmyth K. Genes Dev. 1999; 13: 2039-2058Crossref PubMed Scopus (575) Google Scholar). 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 (12Morgan D.O. Nat. Cell Biol. 1999; 1: E47-E53Crossref PubMed Scopus (306) Google Scholar, 13Zachariae W. Curr. Opin. Cell Biol. 1999; 11: 708-716Crossref PubMed Scopus (41) Google Scholar). Cdc14 drives both of these processes by dephosphorylating at least three targets: Hct1, Swi5, and Sic1 (8Visintin R. Craig K. Hwang E.S. Prinz S. Tyers M. Amon A. Mol. Cell. 1998; 2: 709-718Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar,10Jaspersen S.L. Charles J.F. Morgan D.O. Curr. Biol. 1999; 9: 227-236Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). 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 (8Visintin R. Craig K. Hwang E.S. Prinz S. Tyers M. Amon A. Mol. Cell. 1998; 2: 709-718Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 10Jaspersen S.L. Charles J.F. Morgan D.O. Curr. Biol. 1999; 9: 227-236Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). 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 (8Visintin R. Craig K. Hwang E.S. Prinz S. Tyers M. Amon A. Mol. Cell. 1998; 2: 709-718Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). The Sic1 protein itself may be protected from premature degradation when dephosphorylated by Cdc14 (8Visintin R. Craig K. Hwang E.S. Prinz S. Tyers M. Amon A. Mol. Cell. 1998; 2: 709-718Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). 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. 15Visintin R. Amon A. Curr. Opin. Cell Biol. 2000; 12: 372-377Crossref PubMed Scopus (151) Google Scholar, 16Cockell M.M. Gasser S.M. Curr. Biol. 1999; 9: R575-R576Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 17Garcia S.N. Pillus L. Cell. 1999; 97: 825-828Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). From G1 to anaphase, Net1 sequesters Cdc14 in the nucleolus, where its access to substrate is limited, and its phosphatase activity is suppressed (18Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 19Visintin R. Hwang E.S. Amon A. Nature. 1999; 398: 818-823Crossref PubMed Scopus (488) Google Scholar, 20de Almeida A. Raccurt I. Peyrol S. Charbonneau M. Biol. Cell. 1999; 91: 649-663Crossref PubMed Google Scholar). At late anaphase, Cdc14 is transiently released from Net1 permitting the active phosphatase to reach targets in the nucleus and cytoplasm (18Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 19Visintin R. Hwang E.S. Amon A. Nature. 1999; 398: 818-823Crossref PubMed Scopus (488) Google Scholar). The RENT complex appears to have multiple functions besides Cdc14 regulation including roles in maintaining the integrity of the nucleolus (20de Almeida A. Raccurt I. Peyrol S. Charbonneau M. Biol. Cell. 1999; 91: 649-663Crossref PubMed Google Scholar, 21Straight A.F. Shou W. Dowd G.J. Turck C.W. Deshaies R.J. Johnson A.D. Moazed D. Cell. 1999; 97: 245-256Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar) and sequestering Sir2 to tandem rDNA repeats (21Straight A.F. Shou W. Dowd G.J. Turck C.W. Deshaies R.J. Johnson A.D. Moazed D. Cell. 1999; 97: 245-256Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). 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 (22Guarente L. Genes Dev. 2000; 14: 1021-1026Crossref PubMed Google Scholar, 23Gartenberg M.R. Curr. Opin. Microbiol. 2000; 3: 132-137Crossref PubMed Scopus (91) Google Scholar, 24Gottschling D.E. Curr. Biol. 2000; 10: R708-R711Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (9Jaspersen S.L. Charles J.F. Tinker-Kulberg R.L. Morgan D.O. Mol. Biol. Cell. 1998; 9: 2803-2817Crossref PubMed Scopus (263) Google Scholar, 11Cerutti L. Simanis V. Curr. Opin. Genet. Dev. 2000; 10: 65-69Crossref PubMed Scopus (42) Google Scholar, 12Morgan D.O. Nat. Cell Biol. 1999; 1: E47-E53Crossref PubMed Scopus (306) Google Scholar). 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 (25Bardin A.J. Visintin R. Amon A. Cell. 2000; 102: 21-31Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 26Pereira G. Hofken T. Grindlay J. Manson C. Schiebel E. Mol. Cell. 2000; 6: 1-10Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). 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 (27Cohen-Fix O. Koshland D. Genes Dev. 1999; 13: 1950-1959Crossref PubMed Scopus (103) Google Scholar, 28Shirayama M. Toth A. Galova M. Nasmyth K. Nature. 1999; 402: 203-207Crossref PubMed Scopus (294) Google Scholar, 29Tinker-Kulberg R.L. Morgan D.O. Genes Dev. 1999; 13: 1936-1949Crossref PubMed Scopus (147) Google Scholar). 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 (28Shirayama M. Toth A. Galova M. Nasmyth K. Nature. 1999; 402: 203-207Crossref PubMed Scopus (294) Google Scholar). 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 (25Bardin A.J. Visintin R. Amon A. Cell. 2000; 102: 21-31Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 26Pereira G. Hofken T. Grindlay J. Manson C. Schiebel E. Mol. Cell. 2000; 6: 1-10Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 28Shirayama M. Toth A. Galova M. Nasmyth K. Nature. 1999; 402: 203-207Crossref PubMed Scopus (294) Google Scholar, 29Tinker-Kulberg R.L. Morgan D.O. Genes Dev. 1999; 13: 1936-1949Crossref PubMed Scopus (147) Google Scholar, 30Wang Y. Hu F. Elledge S.J. Curr. Biol. 2000; 10: 1379-1382Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). 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. Plasmids were constructed using standard cloning and polymerase chain reaction techniques as briefly outlined below. The authenticity of all plasmids was verified by DNA sequencing. For two-hybrid analyses, the coding sequence of theCDC14 gene was cloned into the pGBDU-C1 vector (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) to create pGBDU-CDC14, which encodes a fusion protein with theGAL4 DNA binding domain. Using polymerase chain reaction, the NET1 coding sequence was inserted into pGAD-C1 (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) to produce pGAD-NET1, which expresses a fusion protein with theGAL4 activation domain. Restriction fragments derived from pGAD-NET1 were used to construct a series of six pGAD plasmids encoding fusion proteins comprised of Net1 truncation mutants containing residues 1–601, 1–341, 1–207, 92–341, 342–1189, and 92–1189. A pGAD vector encoding a fusion protein with Net1(1–146) was constructed using a fragment amplified by polymerase chain reaction. The completeSWI5 coding sequence and codons 1–600 of NET1were amplified by polymerase chain reaction and inserted into pET21a to generate the pET-SWI5-His6 and pET-NET1(1–600)-His6 expression plasmids. A restriction fragment from pGAD-Net1(1–341) was inserted into pET21a to give pET-NET1(1–341)-His6 expression plasmid. All of these pET expression plasmids encode proteins bearing NH2-terminal T7 tags. A two-hybrid screen was performed by transforming the yeast strain PJ69–4A (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) with the pGBDU-CDC14 plasmid expressing the Cdc14 bait protein. This strain was then transformed with each of three S. cerevisiae Y2HL genomic libraries (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar), and about 2 × 106 colonies with each library were screened for activation of the HIS3and ADE2 reporter genes (see below). After establishing that reporter activation was dependent on the presence of both bait and target proteins, plasmids from positive clones were isolated and sequenced. This screen identified several positive clones containing 5′-coding sequences from the yeast YJL076W (NET1) gene. Two-hybrid assays were used to assess the ability of Net1 truncation mutants to interact with Cdc14. After transformation of the yeast strain PJ69–4A (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) with the pGBDU-CDC14 plasmid and a pGAD vector encoding one of the Net1 truncation mutants (see above) cells were plated on medium permitting detection of reporter gene activation. Ade+ cells were identified by growth on SC medium lacking adenine, leucine, and uracil, whereas His+ cells grew on SC medium lacking histidine, leucine, and uracil but containing 1 mm 3-aminotriazole. Activation of the lacZreporter gene was assessed by performing liquid β-galactosidase assays as described (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Yeast GST-Cdc14 and GST-Cdc14(1–374) were overexpressed in Escherichia coliBL21 (DE3) cells as described (1Taylor G.S. Liu Y. Baskerville C. Charbonneau H. J. Biol. Chem. 1997; 272: 24054-24063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) except that lysates were treated with polyethyleneimine (PEI) prior to affinity purification. Using a stock solution of 5% (w/v) PEI at pH 8, lysates were adjusted to a final PEI concentration of 0.15% (w/v) and centrifuged at 10,000 ×g for 10 min at 4 °C. The pellet was resuspended in 1 volume of lysis buffer containing 500 mm NaCl, mixed and clarified by centrifugation (10,000 × g for 10 min) prior to purification using glutathione-Sepharose (1Taylor G.S. Liu Y. Baskerville C. Charbonneau H. J. Biol. Chem. 1997; 272: 24054-24063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The GST-HCdc14A (1) and GST-HCdc14B (1) fusion proteins were expressed inE. coli BL21 (DE3) cells from pET-GST vectors (1Taylor G.S. Liu Y. Baskerville C. Charbonneau H. J. Biol. Chem. 1997; 272: 24054-24063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and purified from PEI-treated extracts as described above. The TC45 splice variant of the human T cell phosphatase was expressed and purified using the method of Hao et al. (32Hao L. Tiganis T. Tonks N.K. Charbonneau H. J. Biol. Chem. 1997; 272: 29322-29329Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Purified recombinant VHR and PPT1 protein phosphatases were provided by Drs. Zhong-Yin Zhang and S. Rossie, respectively. E. coli BL21 (DE3) cells transformed with expression plasmids encoding Net1(1–600)-His6, Net1(1–341)-His6, and Swi5-His6 were grown at 37 °C in LB medium containing 100 μg/ml ampicillin until theA 600 was about 0.7. Following the addition of 50 μm isopropyl-1-thio-β-d-galactopyranoside, cells expressing Net1(1–600)-His6 and Swi5-His6 were grown for 14–16 h at room temperature, whereas cells producing Net1(1–341)-His6 were induced with 400 μmisopropyl-1-thio-β-d-galactopyranoside and grown for 4 h at 30 °C. Swi5-His6 and Net1(1–600)-His6 were purified directly from extracts using His-Bind resin (Novagen) according to protocols from the manufacturer, whereas lysates containing Net1(1–341)-His6were treated with PEI as described above before purification on His-Bind resin. For further purification, Net1(1–600)-His6 (1–2 mg) was dialyzed with buffer A (50 mm Tris, pH 8.0, 2 mm EDTA, 0.1% (v/v) 2-mercaptoethanol), loaded on a Mono Q HR 5/5 column, and eluted with a 25-ml linear gradient from 0 to 500 mm NaCl in buffer A using a fast protein liquid chromatography system (Amersham Pharmacia Biotech). Peak fractions were combined, dialyzed with 50 mm imidazole, pH 6.6, 1 mm EDTA, 0.1% (v/v) 2-mercapthoethanol for phosphatase assays or with 25 mm Tris, pH 7.4, 2.6 mm KCl, 137 mm NaCl, 0.1% (v/v) 2-mercaptoethanol for in vitro binding assays and concentrated with Centricon-10 (Amicon) membranes. Net1(1–341)-His6 was purified further on a Sephacryl S300 HR 1 × 80-cm column (Amersham Pharmacia Biotech) equilibrated in 50 mm imidazole, pH 6.6, 350 mmNaCl, 1 mm EDTA, 0.1% (v/v) 2-mercapthoethanol and eluted at a flow rate of 0.5 ml/min using the fast protein liquid chromatography system. Peak fractions were combined and concentrated by either ultrafiltration with a Centriprep-10 (Amicon) membrane or dehydration in a dialysis bag covered with dry Sephadex G-25. Sic1-Myc-His6 was expressed as a fusion protein with maltose-binding protein and purified as described (33Verma R. Annan R.S. Huddleston M.J. Carr S.A. Reynard G. Deshaies R.J. Science. 1997; 278: 455-460Crossref PubMed Scopus (392) Google Scholar). The Cdc28 cyclin-dependent kinase was immunopurified from yeast as a complex with either the Clb2 or Cln2 cyclin carrying epitope tags derived from human influenza virus hemagglutinin (HA) as described (34Futcher B. Fantes P. Brooks R. The Cell Cycle: A Practical Approach. Oxford University Press, New York1993: 80-84Google Scholar). Yeast with copies of either GAL1-HA-CLB2 orGAL1-HA-CLN2 genes were grown in medium containing 2% galactose to induce expression and disrupted with glass beads. The HA-Clb2·Cdc28 and HA-Cln2·Cdc28 kinase complexes were immunopurified from extracts using 12CA5 monoclonal antibodies cross-linked to protein A-Sepharose beads (34Futcher B. Fantes P. Brooks R. The Cell Cycle: A Practical Approach. Oxford University Press, New York1993: 80-84Google Scholar). GST, GST-Cdc14, and GST-Cdc14 C283S affinity matrices were prepared by adding 0.5 nmol of each protein to 200 μl of a 50% slurry of glutathione-Sepharose in binding buffer B (25 mm Tris, pH 7.4, 137 mm NaCl, 2.6 mm KCl, 0.1% (v/v) 2-mercaptoethanol). Each mixture was incubated for 30 min at 4 °C in a final volume of 1 ml buffer B. The supernatant was removed, and each of the three affinity matrices was suspended in buffer B and split into two aliquots of equal volume. 1 nmol of Net1 in buffer B was added to one aliquot of each affinity matrix (final volume of 1 ml of buffer B), whereas the second sample of affinity resin received an equal volume of buffer B without Net1. After mixing by inversion for 30 min at 4 °C, the resin was centrifuged and washed four times with 1 ml of buffer B containing 0.01% (v/v) Triton X-100. Washed beads were suspended in 50 μl of 2 × SDS-PAGE loading buffer, boiled for 5 min, and an aliquot (20 μl) was separated on a 12% SDS-polyacrylamide gel. Proteins were visualized by staining the gel with Coomassie Blue. Densitometry was used to estimate the relative amounts of Net1 fragment and GST-Cdc14 proteins in the SDS gels assuming that the two proteins have equal staining with Coomassie dye. Images of gels dried between translucent membranes were obtained from a scanner (Astra 2200, Umax) used in the transparency mode and analyzed using ImageQuant software (Molecular Dynamics). Swi5 and histone H1 were phosphorylated with [γ-32P]ATP using the partially purified HA-Clb2·Cdc28 complex, whereas Sic1 was phosphorylated with the HA-Cln2·Cdc28 kinase (see above). Myelin basic protein (MBP) (Life Technologies, Inc.) was phosphorylated on tyrosine using GST-lyn kinase as described (35Sheng Z. Charbonneau H. J. Biol. Chem. 1993; 268: 4728-4733Abstract Full Text PDF PubMed Google Scholar). Radiolabeled casein phosphorylated on Ser residues was provided by Dr. S. Rossie. Protein substrate concentrations given herein represent the total concentration of phosphorylated residues used in each assay. The phosphatase activity of yeast Cdc14, HCdc14A, HCdc14B, VHR, and TC45 toward pNPP and protein substrates was measured as described (1Taylor G.S. Liu Y. Baskerville C. Charbonneau H. J. Biol. Chem. 1997; 272: 24054-24063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 32Hao L. Tiganis T. Tonks N.K. Charbonneau H. J. Biol. Chem. 1997; 272: 29322-29329Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) in reactions carried out at 30 °C for 5–40 min in buffer containing 50 mm imidazole, pH 6.6, 1 mm EDTA, 1 mm dithiothreitol, and 0.5 mg/ml bovine serum albumin. Assays with radiolabeled Sic1, Swi5, histone H1, and Tyr(P)-MBP substrates were performed in a total volume of 30 μl; 50-μl reactions were employed for pNPP and casein. The final assay buffer for yeast PPT1 contained 56 mm Tris, 36 mm imidazole, pH 7.2, 1.8 mm EDTA, 1.2 mm EGTA, 0.1% 2-mercaptoethanol, 0.5 mg/ml bovine serum albumin, and 6 μm phosphocasein. Digestion with Staphylococcus aureus V8 protease (0.15 μg) was performed for 60 min at room temperature with 75.5 μg Net1(1–600) in 0.5 ml of buffer containing 40 mm ammonium bicarbonate, pH 7.8, 4 mm Tris, 22 mm NaCl, 0.4 mm KCl, 3% (v/v) glycerol, and 0.02% (v/v) 2-mercaptoethanol. Cleavage with 1.5 μg of endoproteinase Arg-C (stored at −20 °C in water) was carried out at 30 °C as described above for staphylococcal V8 protease except that the digestion buffer contained 80 mmammonium bicarbonate at pH 8. Aliquots (50 μl) of each digest were precipitated with trichloroacetic acid as described previously (32Hao L. Tiganis T. Tonks N.K. Charbonneau H. J. Biol. Chem. 1997; 272: 29322-29329Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and analyzed on 12% SDS-polyacrylamide gels. The remainder of each reaction (equivalent to 1 nmol of the Net1(1–600) fragment) was stopped by adding soybean trypsin inhibitor to a final concentration of 1 mg/ml and used for in vitro binding assays with affinity matrices containing 0.25 nmol of GST-Cdc14 or GST (see above). For amino acid sequencing, proteolytic fragments were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes as described (36Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). Protein bands on the membrane were stained with Coomassie Blue, excised, and analyzed by automated gas phase sequencing. Because of its high affinity for Cdc14, the Henderson method (37Henderson P.J. Biochem. J. 1972; 127: 321-333Crossref PubMed Scopus (478) Google Scholar) for tight binding inhibitors was used for the analysis of Net1 inhibition. For these experiments, the activity of GST-Cdc14 (5 nm) was measured at six different substrate concentrations ranging from 0.27 to 17 μm Tyr(P)-MBP. For each concentration of substrate, six assays were performed with Net1(1–600) concentrations between 0 and 100 nm. For each substrate concentration, the value It/(1 −v i/v 0) was plotted on they axis versus v 0/v i on the xaxis, where It is the total Net1(1–600) concentration, andv i and v 0 are the velocities with and without inhibitor, respectively. This plot generates a series of lines for each concentration of substrate which were fit by unweighted linear regression using a fixed intercept value of 5 nm, which was the total enzyme concentration (37Henderson P.J. Biochem. J. 1972; 127: 321-333Crossref PubMed Scopus (478) Google Scholar). TheK i and mechanism of inhibition were determined from a replot of the slopes of these lines versus substrate concentration. In addition to a genetic screen described by Shou et al. (18Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar), we and others (19Visintin R. Hwang E.S. Amon A. Nature. 1999; 398: 818-823Crossref PubMed Scopus (488) Google Scholar) also identified Net1 in a yeast two-hybrid screen for regulators or substrates of Cdc14 (TableI). Along with the immunolocalization and genetic studies (18Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 19Visintin R. Hwang E.S. Amon A. Nature. 1999; 398: 818-823Crossref PubMed Scopus (488) Google Scholar, 20de Almeida A. Raccurt I. Peyrol S. Charbonneau M. Biol. Cell. 1999; 91: 649-663Crossref PubMed Google Scholar), these two-hybrid results support the notion that Net1 and Cdc14 interact in vivo, but they fail to establish whether this interaction is direct or mediated by other proteins. To determine whether there is a direct interaction between Net1 and Cdc14 in vitro, we used binding assays employing recombinant GST-Cdc14 (1Taylor G.S. Liu Y. Baskerville C. Charbonneau H. J. Biol. Chem. 1997; 272: 24054-24063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and Net1(1–600)-His6. This NH2-terminal fragment of Net1 was used to preclude problems associated with the bacterial expression of the 1,189-residue full-length protein. Sequence analysis of Net1 clones obtained in our two-hybrid screens indicated that the NH2-terminal half of Net1 (residues 1–641) interacted with Cdc14. A two-hybrid assay confirmed that a fragment of Net1 containing residues 1–601 could interact with Cdc14 (Table I, third row).Table ITwo-hybrid interaction of Net1 or Net1 mutants with Cdc14Activation domain fusion proteinADE2activationHIS3activation1-aTransformants were screened for activation of the HIS3 gene on SC-His-Leu-uracil medium containing 1 mm 3-aminotriazole.LacZactivationβ-Gal activity1-bβ-Galactosidase activities are the mean of values obtained in triplicate. These data are representative of the results obtained from four different experiments.Fold activation1-cValues differing from control by more than 0.5 Miller unit were statistically significant indicating activation of the lacZ gene as shown with a plus (+) sign.Miller unitspGAD-C3 alone−−3.21.0 /−Net1+−4.51.4 /+Net1(1–601)++12.43.8 /+Net1(1–341)++8.02.5 /+Net1(342–1189)−−3.11.0 /−Net1(1–207)+−3.51.1 /−Net1(1–146)−−2.10.6 /−Net1(92–1189)++4.51.4 /+Net1(92–341)−−3.11.0 /−Yeast two-hybrid assays (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) were used to assess the ability of Net1 or Net1 truncation mutants to interact with Cdc14. Yeast (PJ69–4A) expressing a Cdc14 fusion protein with the GAL4 DNA binding domain (pGBDU-CDC14) were transformed with pGAD plasmids encoding Net1 or Net1 truncation mut
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