PR48, a Novel Regulatory Subunit of Protein Phosphatase 2A, Interacts with Cdc6 and Modulates DNA Replication in Human Cells

ABSTRACT Initiation of DNA replication in eukaryotes is dependent on the activity of protein phosphatase 2A (PP2A), but specific phosphoprotein substrates pertinent to this requirement have not been identified. A novel regulatory subunit of PP2A, termed PR48, was identified by a yeast two-hybrid screen of a human placental cDNA library, using human Cdc6, an essential component of prereplicative complexes, as bait. PR48 binds specifically to an amino-terminal segment of Cdc6 and forms functional holoenzyme complexes with A and C subunits of PP2A. PR48 localizes to the nucleus of mammalian cells, and its forced overexpression perturbs cell cycle progression, causing a G1 arrest. These results suggest that dephosphorylation of Cdc6 by PP2A, mediated by a specific interaction with PR48, is a regulatory event controlling initiation of DNA replication in mammalian cells.

[1]  S. Forsburg,et al.  Coupling DNA replication to the cell cycle. , 1993, Cold Spring Harbor symposia on quantitative biology.

[2]  S. Bell,et al.  The multidomain structure of Orc1 p reveals similarity to regulators of DNA replication and transcriptional silencing , 1995, Cell.

[3]  R. Bassel-Duby,et al.  Transient expression of a winged-helix protein, MNF-beta, during myogenesis , 1997, Molecular and cellular biology.

[4]  T. Coleman,et al.  The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts , 1996, Cell.

[5]  P. Russell,et al.  Negative regulation of Cdc18 DNA replication protein by Cdc2. , 1998, Molecular biology of the cell.

[6]  R. Laskey,et al.  MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells , 1995, Nature.

[7]  T. Hunter,et al.  Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  G. Williams,et al.  Improved cervical smear assessment using antibodies against proteins that regulate DNA replication. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Mumby,et al.  Diversity in the regulatory B-subunits of protein phosphatase 2A: identification of a novel isoform highly expressed in brain. , 1994, Biochemistry.

[10]  M. Mumby,et al.  Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen , 1991, Molecular and cellular biology.

[11]  K. Scheidtmann,et al.  Protein phosphatase 2A is required for the initiation of chromosomal DNA replication. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Parvin,et al.  Human CDC6/Cdc18 Associates with Orc1 and Cyclin-cdk and Is Selectively Eliminated from the Nucleus at the Onset of S Phase , 1998, Molecular and Cellular Biology.

[13]  J. Li,et al.  Ectopic induction of Clb2 in early G1 phase is sufficient to block prereplicative complex formation in Saccharomyces cerevisiae. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  P. Nurse,et al.  p65 cdc18 Plays a major role controlling the initiation of DNA replication in fission yeast , 1995, Cell.

[15]  M. Mumby,et al.  Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. , 1994, The Journal of biological chemistry.

[16]  K. Stoeber,et al.  Cdc6 protein causes premature entry into S phase in a mammalian cell‐free system , 1998, The EMBO journal.

[17]  L. Hartwell.  Sequential function of gene products relative to DNA synthesis in the yeast cell cycle. , 1976, Journal of molecular biology.

[18]  P. Jallepalli,et al.  sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  R V Shohet,et al.  A human protein related to yeast Cdc6p. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Bruce Stillman,et al.  Smart machines at the DNA replication fork , 1994, Cell.

[21]  B. Stillman,et al.  Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin. , 1998, Science.

[22]  K. Arndt,et al.  The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis. , 1995, The EMBO journal.

[23]  M. Mumby,et al.  Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. , 1993, Physiological reviews.

[24]  R. Bernards,et al.  Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein , 1999, Oncogene.

[25]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[26]  P. Jallepalli,et al.  Interaction of the S phase regulator cdc18 with cyclin-dependent kinase in fission yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Nevins,et al.  E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. , 1998, Genes & development.

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

[29]  J. Nevins,et al.  Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Blow,et al.  Chromatin proteins involved in the initiation of DNA replication. , 1997, Current opinion in genetics & development.

[31]  S. Bell,et al.  Initiation of DNA replication in eukaryotic cells. , 1997, Annual review of cell and developmental biology.

[32]  S. Shaffer,et al.  Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication , 1994, Molecular and cellular biology.

[33]  M. Mumby,et al.  Identification of a Novel Protein Phosphatase 2A Regulatory Subunit Highly Expressed in Muscle (*) , 1996, The Journal of Biological Chemistry.

[34]  D. Virshup,et al.  Identification of a New Family of Protein Phosphatase 2A Regulatory Subunits (*) , 1995, The Journal of Biological Chemistry.

[35]  T. Kelly,et al.  cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[36]  B. Hemmings,et al.  Serine/threonine protein phosphatases. , 1995, The Biochemical journal.

[37]  J. Hofsteenge,et al.  Structure and expression of a 72-kDa regulatory subunit of protein phosphatase 2A. Evidence for different size forms produced by alternative splicing. , 1993, The Journal of biological chemistry.

[38]  M. Milán,et al.  Regulation of LIM homeodomain activity in vivo: a tetramer of dLDB and apterous confers activity and capacity for regulation by dLMO. , 1999, Molecular cell.

[39]  B. Stillman,et al.  Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. , 1997, Genes & development.

[40]  R. Fotedar,et al.  Cell cycle control of DNA replication. , 1995, Progress in cell cycle research.

[41]  Bruce Stillman,et al.  ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome , 1995, Cell.

[42]  B. Stillman,et al.  Conserved Initiator Proteins in Eukaryotes , 1995, Science.

[43]  K Nasmyth,et al.  Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a ‘reductional’ anaphase in the budding yeast Saccharomyces cerevisiae. , 1995, The EMBO journal.

[44]  Jiri Bartek,et al.  Phosphorylation of mammalian CDC6 by Cyclin A/CDK2 regulates its subcellular localization , 1999, The EMBO journal.