The Activity of Cdc14p, an Oligomeric Dual Specificity Protein Phosphatase from Saccharomyces cerevisiae, Is Required for Cell Cycle Progression*

The essential CDC14 gene of the budding yeast, Saccharomyces cerevisiae, encodes a 62-kDa protein containing a sequence that conforms to the active site motif found in all enzymes of the protein tyrosine phosphatase superfamily. Genetic studies suggest that Cdc14p may be involved in the initiation of DNA replication, but its precise cell cycle function is unknown. Recombinant Cdc14p was produced in bacteria, characterized, and shown to be a dual specificity protein phosphatase. Polyanions such as polyglutamate and double-stranded and single-stranded DNA bind to Cdc14p and affect its activity. Native molecular weights of 131,000 and 169,000 determined by two independent methods indicate that recombinant Cdc14p self-associates in vitro to form active oligomers. The catalytically inactive Cdc14p C283S/R289A mutant is not able to suppress the temperature sensitivity of acdc14–1 ts mutant nor replace the wild type genein vivo, demonstrating that phosphatase activity is required for the cell cycle function of Cdc14p. A distinctive COOH-terminal segment (residues 375–551) is rich in Asn and Ser residues, carries a net positive charge, and contains two tandem 21-residue repeats. This COOH-terminal segment is not required for activity, for oligomerization, or for the critical cell cycle function of Cdc14p.

[1]  E. Krebs,et al.  [43] Preparation of homogeneous cyclic AMP-dependent protein kinase(s) and its subunits from rabbit skeletal muscle , 1974 .

[2]  H. Xu,et al.  CDC14 of Saccharomyces cerevisiae. Cloning, sequence analysis, and transcription during the cell cycle. , 1992, The Journal of biological chemistry.

[3]  J. Denu,et al.  Purification and Kinetic Characterization of the Mitogen-activated Protein Kinase Phosphatase rVH6* , 1996, The Journal of Biological Chemistry.

[4]  J. Dixon,et al.  New vectors for high level expression of recombinant proteins in bacteria. , 1992, Analytical biochemistry.

[5]  A. Cigan,et al.  Sequence and structural features associated with translational initiator regions in yeast--a review. , 1987, Gene.

[6]  B. Neel,et al.  From Form to Function: Signaling by Protein Tyrosine Phosphatases , 1996, Cell.

[7]  P. Berg,et al.  Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein , 1995, Molecular and cellular biology.

[8]  Ellson Y. Chen,et al.  Overview of manual and automated DNA sequencing by the dideoxy chain termination method , 1991 .

[9]  H. Charbonneau,et al.  The baculovirus Autographa californica encodes a protein tyrosine phosphatase. , 1993, The Journal of biological chemistry.

[10]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[11]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[12]  R. Rothstein.  One-step gene disruption in yeast. , 1983, Methods in enzymology.

[13]  E. Fauman,et al.  Structure and function of theprotein tyrosine phosphatases , 1996 .

[14]  J. Noel,et al.  Structural basis for inhibition of receptor protein-tyrosine phosphatase-α by dimerization , 1996, Nature.

[15]  D. Koshland,et al.  Addition of extra origins of replication to a minichromosome suppresses its mitotic loss in cdc6 and cdc14 mutants of Saccharomyces cerevisiae. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  G. Ashwell,et al.  Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins. , 1976, The Journal of biological chemistry.

[17]  Y. Nishizuka,et al.  Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase. , 1985, The Journal of biological chemistry.

[18]  J. E. Kranz,et al.  Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Yamazaki,et al.  Fifteen open reading frames in a 30·8 kb region of the right arm of chromosome VI from Saccharomyces cerevisiae , 1996, Yeast.

[20]  C. Der,et al.  The Mitogen-activated Protein Kinase Phosphatases PAC1, MKP-1, and MKP-2 Have Unique Substrate Specificities and Reduced Activity in Vivo toward the ERK2 sevenmaker Mutation (*) , 1996, The Journal of Biological Chemistry.

[21]  J. R. Johnston.  Molecular genetics of yeast :a practical approach , 1994 .

[22]  M. Cobb,et al.  Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. , 1993, The Journal of biological chemistry.

[23]  J. Rine,et al.  The origin recognition complex in silencing, cell cycle progression, and DNA replication. , 1995, Molecular biology of the cell.

[24]  P. Russell,et al.  p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast. , 1991, The EMBO journal.

[25]  J. Dixon,et al.  Form and Function in Protein Dephosphorylation , 1996, Cell.

[26]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[27]  L. Hartwell,et al.  A dependent pathway of gene functions leading to chromosome segregation in Saccharomyces cerevisiae , 1982, The Journal of cell biology.

[28]  K Nasmyth,et al.  Viewpoint: Putting the Cell Cycle in Order , 1996, Science.

[29]  C. F. Hardy.  Characterization of an essential Orc2p-associated factor that plays a role in DNA replication , 1996, Molecular and cellular biology.

[30]  J. Diffley,et al.  Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells. , 1996, Genes & development.

[31]  D. Schild,et al.  Diploid spore formation and other meiotic effects of two cell-division-cycle mutations of Saccharomyces cerevisiae. , 1980, Genetics.

[32]  D. Barford,et al.  Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. , 1995, Science.

[33]  D. Barford,et al.  Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Dixon,et al.  Kinetic Analysis of the Catalytic Domain of Human Cdc25B* , 1996, The Journal of Biological Chemistry.

[35]  P. Hieter,et al.  Establishing genetic interactions by a synthetic dosage lethality phenotype. , 1996, Genetics.

[36]  J. Dixon,et al.  The Purification and Characterization of a Human Dual-specific Protein Tyrosine Phosphatase (*) , 1995, The Journal of Biological Chemistry.

[37]  S. Keyse.  An emerging family of dual specificity MAP kinase phosphatases. , 1995, Biochimica et biophysica acta.

[38]  B. Stillman,et al.  Cell Cycle Control of DNA Replication , 1996, Science.

[39]  D. Freifelder.  [9] Zonal centrifugation , 1973 .

[40]  J. Dixon,et al.  Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. , 1991, Analytical biochemistry.

[41]  J. Nickoloff,et al.  Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. , 1992, Analytical biochemistry.

[42]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[43]  J. Olmsted,et al.  Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. , 1981, The Journal of biological chemistry.