title: Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1.
abstract: Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.
authors.affiliation: Department of Cancer Biology, The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: S
authors.initials: S
authors.lastname: Benzeno
authors.forename: F
authors.initials: F
authors.lastname: Lu
authors.forename: M
authors.initials: M
authors.lastname: Guo
authors.forename: O
authors.initials: O
authors.lastname: Barbash
authors.forename: F
authors.initials: F
authors.lastname: Zhang
authors.forename: J G
authors.initials: JG
authors.lastname: Herman
authors.forename: P S
authors.initials: PS
authors.lastname: Klein
authors.forename: A
authors.initials: A
authors.lastname: Rustgi
authors.forename: J A
authors.initials: JA
authors.lastname: Diehl
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D000818:Animals; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D056741:Cyclin D; D019938:Cyclin D1 / Q000235:genetics* / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera
chemical_list: D056741:Cyclin D; D016213:Cyclins; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D010769:Phosphothreonine; D019938:Cyclin D1; D000071679:Glycogen Synthase Kinase 3 beta; C495900:CDK4 protein, human; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D038362:Glycogen Synthase Kinase 3