RanBP2 and SENP3 function in a mitotic SUMO2/3 conjugation-deconjugation cycle on Borealin.
ABSTRACT: The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation-deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.
Project description:The ubiquitin-like SUMO system functions by a cyclic process of modification and demodification, and recent data suggest that the nucleolus is a site of sumoylation-desumoylation cycles. For example, the tumour suppressor ARF stimulates sumoylation of nucleolar proteins. Here, we show that the nucleolar SUMO-specific protease SENP3 is associated with nucleophosmin (NPM1), a crucial factor in ribosome biogenesis. SENP3 catalyses desumoylation of NPM1-SUMO2 conjugates in vitro and counteracts ARF-induced modification of NPM1 by SUMO2 in vivo. Intriguingly, depletion of SENP3 by short interfering RNA interferes with nucleolar ribosomal RNA processing and inhibits the conversion of the 32S rRNA species to the 28S form, thus phenocopying the processing defect observed on depletion of NPM1. Moreover, mimicking constitutive modification of NPM1 by SUMO2 interferes with 28S rRNA maturation. These results define SENP3 as an essential factor for ribosome biogenesis and suggest that deconjugation of SUMO2 from NPM1 by SENP3 is critically involved in 28S rRNA maturation.
Project description:The chromosomal passenger complex (CPC) localizes to centromeres where it activates the mitotic checkpoint in response to inappropriate inter-kinetochore tension. This error correction function is essential for proper chromosome segregation. Here we define several critical features of CPC localization and function. First, the Borealin dimerization domain suppresses dynamic exchange at the centromere to allow optimal CPC function. Second, Borealin dimerization is essential to target a subpopulation of CPC proximal to the kinetochore when the mitotic spindle is disrupted. This subpopulation is also needed for full CPC checkpoint function. The existence of a pool of CPC at the kinetochore suggests that error correction is more complicated than predicted from the Aurora B phosphorylation gradient model. Finally, Haspin kinase plays a key role in maintaining the slowly exchanging centromere Borealin pool, while Aurora B and Mps1 play minimal roles in maintaining CPC localization once cells are in mitosis.
Project description:The chromosomal passenger complex (CPC) comprises at least four protein components and functions at various cellular localizations during different mitotic stages to ensure correct chromosome segregation and completion of cytokinesis. Borealin, the most recently identified member of the CPC, is an intrinsically unstructured protein of low solubility and stability. Recent reports have demonstrated the formation of binary or ternary CPC subcomplexes incorporating short Borealin fragments in vitro. Using isothermal titration calorimetry, we show that full-length Borealin, instead of a Borealin fragment possessing the complete Survivin and INCENP recognition sequence, is required for the composition of a Borealin-Survivin complex competent to interact with INCENP. In addition, we show evidence that full-length Borealin, which forms high-order oligomers in its isolated form, is a monomer in the Borealin-Survivin CPC subcomplex.
Project description:Mixed lineage leukemia 5 (MLL5) is a versatile nuclear protein associated with many cellular events. We have shown previously that phosphorylation of MLL5 by Cdk1 is required for mitotic entry. In this paper, the function of MLL5 in mitotic regulation is further explored. SiRNA-mediated downregulation of MLL5 caused improper chromosome alignment at metaphase and resulted in failure of DNA segregation and cytokinesis. Mechanistic studies revealed that the chromosomal passenger complex (CPC), which plays a key role in chromosomal bi-orientation, was delocalized from the inner centromere region because of proteasome-mediated degradation in MLL5-depleted cells. Biochemical analyses further demonstrated that the central domain of MLL5 interacted with the C-terminus of Borealin, and the interaction is essential to maintain the stability of Borealin. Moreover, the mitotic defects in MLL5-depleted cells were rescued by overexpression of FLAG-MLL5, but not by a FLAG-MLL5 mutant that did not contain the central domain. Collectively, our results suggest that MLL5 functionally interacts with Borealin, facilitates the expression of CPC, and hence contributes to mitotic fidelity and genomic integrity.
Project description:Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.
Project description:Congenital hypothyroidism is the most common neonatal endocrine disorder and is primarily caused by developmental abnormalities otherwise known as thyroid dysgenesis (TD). We performed whole exome sequencing (WES) in a consanguineous family with TD and subsequently sequenced a cohort of 134 probands with TD to identify genetic factors predisposing to the disease. We identified the novel missense mutations p.S148F, p.R114Q and p.L177W in the BOREALIN gene in TD-affected families. Borealin is a major component of the Chromosomal Passenger Complex (CPC) with well-known functions in mitosis. Further analysis of the missense mutations showed no apparent effects on mitosis. In contrast, expression of the mutants in human thyrocytes resulted in defects in adhesion and migration with corresponding changes in gene expression suggesting others functions for this mitotic protein. These results were well correlated with the same gene expression pattern analysed in the thyroid tissue of the patient with BOREALIN-p.R114W. These studies open new avenues in the genetics of TD in humans.
Project description:Acute promyelocytic leukemia is characterized by a chromosomal translocation that produces an oncogenic fusion protein of the retinoic acid receptor ? (RAR?) and promyelocytic leukemia protein (PML). Arsenic trioxide chemotherapy of this cancer induces the PML moiety to organize nuclear bodies, where the oncoprotein is degraded. This process requires the participation of two SUMO paralogs (SUMO1 and SUMO2) to promote PML ubiquitylation mediated by the ubiquitin E3 ligase RNF4 and reorganization of PML nuclear bodies. We demonstrated that the ubiquitylation of PML required the SUMO deconjugation machinery, primarily the deconjugating enzyme SENP1, and was suppressed by expression of non-deconjugatable SUMO2. We hypothesized that constitutive SUMO2 conjugation and deconjugation occurred basally and that arsenic trioxide treatment caused the exchange of SUMO2 for SUMO1 on a fraction of Lys(65) in PML. On the basis of data obtained with mutational analysis and quantitative proteomics, we propose that the SUMO switch at Lys(65) of PML enhanced nuclear body formation, subsequent SUMO2 conjugation to Lys(160), and consequent RNF4-dependent ubiquitylation of PML. Our work provides insights into how the SUMO system achieves selective SUMO paralog modification and highlights the crucial role of SENPs in defining the specificity of SUMO signaling.
Project description:Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1 (HP1) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1. This borealin-HP1 interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1 interaction, demonstrating the spatial requirement of HP1 in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1 and CPC localization in the centromere and illustrate the critical role of borealin-HP1 interaction in orchestrating an accurate cell division.
Project description:The chromosomal passenger complex (CPC) regulates various events in cell division. This complex is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin. Together, these four subunits interdependently regulate CPC function, and they are highly conserved among eukaryotes. However, a Borealin homologue has never been characterized in the fission yeast, Schizosaccharomyces pombe. Here, we isolate a previously uncharacterized S. pombe protein through association with the Cdc14 phosphatase homologue, Clp1/Flp1, and identify it as a Borealin-like member of the CPC. Nbl1 (novel Borealin-like 1) physically associates with known CPC components, affects the kinase activity and stability of the S. pombe Aurora B homologue, Ark1, colocalizes with known CPC subunits during mitosis, and shows sequence similarity to human Borealin. Further analysis of the Clp1-Nbl1 interaction indicates that Clp1 requires CPC activity for proper accumulation at the contractile ring (CR). Consistent with this, we describe negative genetic interactions between mutant alleles of CPC and CR components. Thus, this study characterizes a fission yeast Borealin homologue and reveals a previously unrecognized connection between the CPC and the process of cytokinesis in S. pombe.
Project description:Posttranslational modification with small ubiquitin-like modifier (Sumo) regulates numerous cellular and developmental processes. Sumoylation is dynamic with deconjugation by Sumo-specific proteases (Senps) regulating steady-state levels. Different Senps are found in distinct subcellular domains, which may limit their deconjugation activity to colocalizing Sumo-modified proteins. In vitro, Senps can discriminate between the different Sumo paralogs: Sumo1 versus the highly related Sumo2 and Sumo3 (Sumo2/3), which can form poly-Sumo chains. However, a full understanding of Senp specificity in vivo is still lacking. Here, using biochemical and genetic approaches, we establish that Senp1 has an essential, nonredundant function to desumoylate Sumo1-modified proteins during mouse embryonic development. Senp1 specificity for Sumo1 conjugates represents an intrinsic function and not simply a product of colocalization. In contrast, Senp1 has only a limited role in Sumo2/3 desumoylation, although it may regulate Sumo1-mediated termination of poly-Sumo2/3 chains.