title: Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis.
abstract: The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.
authors.affiliation: Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, Scotland, United Kingdom.
authors.forename: James R A
authors.initials: JR
authors.lastname: Hutchins
authors.forename: William J
authors.initials: WJ
authors.lastname: Moore
authors.forename: Fiona E
authors.initials: FE
authors.lastname: Hood
authors.forename: Jamie S J
authors.initials: JS
authors.lastname: Wilson
authors.forename: Paul D
authors.initials: PD
authors.lastname: Andrews
authors.forename: Jason R
authors.initials: JR
authors.lastname: Swedlow
authors.forename: Paul R
authors.initials: PR
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D018797:Cell Cycle Proteins; D002875:Chromosomes / Q000378:metabolism* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D028884:Karyopherins / Q000378:metabolism; D008164:Luminescent Proteins; D008863:Microspheres; D008938:Mitosis / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured; D014981:Xenopus; D029867:Xenopus Proteins; D020931:ran GTP-Binding Protein / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D028884:Karyopherins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; C120587:RAN protein, human; C499304:RCC1 protein, Xenopus; C062521:RCC1 protein, human; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; D020931:ran GTP-Binding Protein