title: Differential regulation of CDP/Cux p110 by cyclin A/Cdk2 and cyclin A/Cdk1.
abstract: Previous experiments with peptide fusion proteins suggested that cyclin A/Cdk1 and Cdk2 might exhibit similar yet distinct phosphorylation specificities. Using a physiological substrate, CDP/Cux, our study confirms this notion. Proteolytic processing of CDP/Cux by cathepsin L generates the CDP/Cux p110 isoform at the beginning of S phase. CDP/Cux p110 makes stable interactions with DNA during S phase but is inhibited in G2 following the phosphorylation of serine 1237 by cyclin A/Cdk1. In this study, we propose that differential phosphorylation by cyclin A/Cdk1 and cyclin A/Cdk2 enables CDP/Cux p110 to exert its function as a transcriptional regulator specifically during S phase. We found that like cyclin A/Cdk1, cyclin A/Cdk2 interacted efficiently with recombinant CDP/Cux proteins that contain the Cut homeodomain and an adjacent cyclin-binding motif (Cy). In contrast to cyclin A/Cdk1, however, cyclin A/Cdk2 did not efficiently phosphorylate CDP/Cux p110 on serine 1237 and did not inhibit its DNA binding activity in vitro. Accordingly, co-expression with cyclin A/Cdk2 in cells did not inhibit the DNA binding and transcriptional activities of CDP/Cux p110. To confirm that the sequence surrounding serine 1237 was responsible for the differential regulation by Cdk1 and Cdk2, we replaced 4 amino acids flanking the phosphorylation site to mimic a known Cdk2 phosphorylation site present in the Cdc6 protein. Both cyclin A/Cdk2 and Cdk1 efficiently phosphorylated the CDP/Cux(Cdc6) mutant and inhibited its DNA binding activity. Altogether our results help explain why the DNA binding activity of CDP/Cux p110 is maximal during S phase and decreases in G2 phase.
authors.affiliation: Molecular Oncology Group, McGill University Health Center, McGill University, Montreal, Quebec H3A 1A1, Canada.
authors.forename: Marianne
authors.initials: M
authors.lastname: Santaguida
authors.forename: Alain
authors.initials: A
authors.lastname: Nepveu
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D019925:Cyclin A / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D016195:G2 Phase; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics*; D016196:S Phase; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; C000624101:Cux1 protein, mouse; D019925:Cyclin A; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase; D016203:CDC2 Protein Kinase; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse