title: Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation.
abstract: Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.
authors.affiliation: a Institute of Enzymology; RCNS; Hungarian Academy of Sciences ; Budapest , Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Róna
authors.forename: Máté
authors.initials: M
authors.lastname: Borsos
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Ellis
authors.forename: Ahmed M
authors.initials: AM
authors.lastname: Mehdi
authors.forename: Mary
authors.initials: M
authors.lastname: Christie
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Környei
authors.forename: Máté
authors.initials: M
authors.lastname: Neubrandt
authors.forename: Judit
authors.initials: J
authors.lastname: Tóth
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Bozóky
authors.forename: László
authors.initials: L
authors.lastname: Buday
authors.forename: Emília
authors.initials: E
authors.lastname: Madarász
authors.forename: Mikael
authors.initials: M
authors.lastname: Bodén
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
authors.forename: Beáta G
authors.initials: BG
authors.lastname: Vértessy
mesh_terms: D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division; D018844:Cyclin-Dependent Kinases / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D006801:Humans; D019913:Nuclear Localization Signals / Q000235:genetics*; D010766:Phosphorylation; D020543:Proteome
chemical_list: D019913:Nuclear Localization Signals; D020543:Proteome; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases
keywords: Cdc28, cyclin-dependent protein kinase (Cdk) encoded by CDC28; Cdk1, cyclin-dependent kinase 1; GO, gene ontology; NES, nuclear export signal; NLS, nuclear localization signal; SNP, single nucleotide polymorphisms; SV40, Simian virus 40; UBA1, Ubiquitin-activating enzyme E1; UNG2, Human Uracil-DNA glycosylase 2; cNLS, classical nuclear localization signal; cell cycle; dNTP, deoxyribonucleotide triphosphate; dTTP, deoxythymidine triphosphate; dUMP, deoxyuridine monophosphate; dUTP, deoxyuridine triphosphate; dUTPase; importin; phosphorylation; trafficking