RCC1 isoforms differ in their affinity for chromatin, molecular interactions and regulation by phosphorylation

Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
Journal of Cell Science (Impact Factor: 5.43). 11/2007; 120(Pt 19):3436-45. DOI: 10.1242/jcs.009092
Source: PubMed


RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Generation of Ran-GTP by RCC1 on chromatin provides a spatial signal that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. We show that RCC1 is expressed in human cells as at least three isoforms, named RCC1alpha, RCC1beta and RCC1gamma, which are expressed at different levels in specific tissues. The beta and gamma isoforms contain short inserts in their N-terminal regions (NTRs) that are not present in RCC1alpha. This region mediates interaction with chromatin, binds importin alpha3 and/or importin beta, and contains regulatory phosphorylation sites. RCC1gamma is predominantly localised to the nucleus and mitotic chromosomes like RCC1alpha. However, compared to RCC1alpha, RCC1gamma has a greatly reduced interaction with an importin alpha3-beta and a stronger interaction with chromatin that is mediated by the extended NTR. RCC1gamma is also the isoform that is most highly phosphorylated at serine 11 in mitosis. Unlike RCC1alpha, RCC1gamma supports cell proliferation in tsBN2 cells more efficiently when serine 11 is mutated to non-phosphorylatable alanine. Phosphorylation of RCC1gamma therefore specifically controls its function during mitosis. These results show that human RCC1 isoforms have distinct chromatin binding properties, different molecular interactions, and are selectively regulated by phosphorylation, as determined by their different NTRs.

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    • "In addition, three RCC1 splicing isoforms (RCC1, , and ) expressed in human cells differ in their NTD composition and in how their binding to chromatin is affected by Ser 11 phosphorylation (Hood and Clarke, 2007). Because quantitative PCR showed that the phosphorylationsensitive RCC1 isoform (Hood and Clarke, 2007) was the least abundant and that RCC1 was the prevailing isoform (Fig. S2 C), we focused on RCC1 and its methylation. Although immunoblotting with RCC1 -N-trimethylation antibodies (Me3-SPK; Schaner Tooley et al., 2010) showed that, relative to RCC1 protein levels, the Me3-SPK on RCC1 was similar in HeLa and HFF-1 cells (41 ± 7% Me3-SPK HFF-1/HeLa), this comparison was limited by residual reactivity of the Me-3 antibody with nonmethylated RCC1 (Fig. 2 A and Fig. S2 E). "
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    ABSTRACT: Many mitotic factors were shown to be activated by Ran guanosine triphosphatase. Previous studies in Xenopus laevis egg extracts and in highly proliferative cells showed that mitotic chromosomes were surrounded by steep Ran guanosine triphosphate (GTP) concentration gradients, indicating that RanGTP-activated factors promote spindle assembly around chromosomes. However, the mitotic role of Ran in normal differentiated cells is not known. In this paper, we show that although the steep mitotic RanGTP gradients were present in rapidly growing cell lines and were required for chromosome congression in mitotic HeLa cells, the gradients were strongly reduced in slow-growing primary cells, such as HFF-1 fibroblasts. The overexpression of RCC1, the guanine nucleotide exchange factor for Ran, induced steeper mitotic RanGTP gradients in HFF-1 cells, showing the critical role of RCC1 levels in the regulation of mitosis by Ran. Remarkably, in vitro fusion of HFF-1 cells produced cells with steep mitotic RanGTP gradients comparable to HeLa cells, indicating that chromosomal gain can promote mitosis in aneuploid cancer cells via Ran.
    Full-text · Article · Jan 2013 · The Journal of Cell Biology
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    • "The preferred substrate of CDK1 is RCC1g isoform whose binding to chromatin is promoted by phosphorylation directly, rather than through preventing importin a3 binding (Hood and Clarke 2007). Interestingly, RCC1g isoform is prevalent in extracts of human ovaries (Hood and Clarke 2007), suggesting that this phosphorylationsensitive isoform may be prevalent in oocytes. A more recent study showed that rather than by mitotic CDK1 kinase, RCC1 is preferably phosphorylated on its N-terminal tail sites by proapoptotic MST1 and MST2 kinases, which in turn are activated by binding to tumor suppressor protein RASSF1A (Dallol et al. 2009). "
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    ABSTRACT: The maturation of vertebrate oocyte into haploid gamete, the egg, consists of two specialized asymmetric cell divisions with no intervening S-phase. Ran GTPase has an essential role in relaying the active role of chromosomes in their own segregation by the meiotic process. In addition to its conserved role as a key regulator of macromolecular transport between nucleus and cytoplasm, Ran has important functions during cell division, including in mitotic spindle assembly and in the assembly of nuclear envelope at the exit from mitosis. The cellular functions of Ran are mediated by RanGTP interactions with nuclear transport receptors (NTRs) related to importin β and depend on the existence of chromosome-centered RanGTP gradient. Live imaging with FRET biosensors indeed revealed the existence of RanGTP gradient throughout mouse oocyte maturation. NTR-dependent transport of cell cycle regulators including cyclin B1, Wee2, and Cdc25B between the oocyte cytoplasm and germinal vesicle (GV) is required for normal resumption of meiosis. After GVBD in mouse oocytes, RanGTP gradient is required for timely meiosis I (MI) spindle assembly and provides long-range signal directing egg cortex differentiation. However, RanGTP gradient is not required for MI spindle migration and may be dispensable for MI spindle function in chromosome segregation. In contrast, MII spindle assembly and function in maturing mouse and Xenopus laevis eggs depend on RanGTP gradient, similar to X. laevis MII-derived egg extracts.
    Full-text · Article · Jan 2011 · Results and problems in cell differentiation
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    • "RCC1β and RCC1γ have unique inserts after residue 24 which alter the length of their N-terminal tails. In the case of RCC1γ, a 17 amino acid insert stabilises its interaction with chromatin, reduces importin binding and alters its regulation by phosphorylation at serine 11 [21]. "
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    ABSTRACT: Background Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. Results We have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. Conclusions These results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1α with chromatin is promoted by a conformational change in the α-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.
    Full-text · Article · Jun 2010 · BMC Cell Biology
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