Mitotic regulation in higher eukaryotes by Ran and SUMO-1

Mary Dasso, PhD, Head, Section on Cell Cycle Regulation
Kara Lukasiewicz, PhD, Postdoctoral Fellow
Alexei Arnaoutov, PhD, Visiting Fellow
Maiko Furuta, PhD, Visiting Fellow Ram
Kumar Mishra, PhD, Visiting Fellow
Debaditya Mukhopadhyay, PhD, Visiting Fellow
Yonggang Wang, PhD, Visiting Fellow
Hyun-Joo Yoon, PhD, Visiting Fellow
Chawon Yun, PhD, Visiting Fellow
Cheoi-Yong Choi, PhD, Guest Researcher
Maia Ouspenskaia, DVM, Biologist/Technician
Yekaterina Boyarchuk, BA, Graduate Student¹

Our studies focus on two closely linked biochemical pathways that have been implicated in both mitotic regulation and nuclear-cytoplasmic trafficking: the SUMO pathway and the Ran pathway. SUMO proteins constitute a family of ubiquitin-like proteins that become covalently conjugated to cellular targets. Our studies on SUMOylation investigate the physiological roles of ubiquitinlike protease/sentrin-specific protease (Ulp/SENP) family members. Ulps/SENPs play a pivotal role in determination of the spectrum of conjugated species by directly regulating the production of free, conjugatable SUMO proteins and the half-life of conjugated species. The Ran GTPase controls many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly, and cell cycle progression. We are particularly interested in the role(s) of Ran at mitotic kinetochores, where it is essential for regulation of the spindle assembly checkpoint and for assembly of microtubule fibers that attach kinetochores to spindle poles. We are currently focusing on mechanisms that target Ran pathway components to the kinetochore as well as on interactions of Ran pathway components at kinetochores with other proteins that are structural or functional components of the interphase nuclear pore.

SUMO family small ubiquitin-like modifiers in higher eukaryotes

Mukhopadhyay, Ouspenskaia, Wang, Yun, Choi; in collaboration with Yergey, Wilkinson

SUMO proteins are ubiquitin-like modifiers that become covalently attached (conjugated) to other proteins within cells, thereby regulating the behavior of their targets. While budding yeast has a single SUMO, called Smt3p, there are three commonly expressed mammalian SUMO paralogues called SUMO-1, SUMO-2, and SUMO-3. SUMO-2 and SUMO-3 are 96 percent identical while SUMO-1 is approximately 45 percent identical to either SUMO-2 or SUMO-3. (Here, SUMO-2 and SUMO -3 will be collectively called SUMO -2/3 when they cannot be distinguished from each other.) SUMO conjugation targets play essential roles in many processes, including gene expression, cell cycle progression, synthesis and repair of DNA, response to extracellular stimuli, and nucleocytoplasmic transport. Through these targets, SUMO proteins have been implicated in many human diseases, including cancers, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and type I diabetes.

The conjugation pathway for SUMO proteins is similar to the ubiquitin conjugation pathway: SUMO proteins are processed by Ulps/SENPs to reveal a diglycine motif at their C-termini. After processing, SUMO proteins undergo ATP-dependent formation of a thioester bond to their activating (E1) enzyme, Aos1/Uba2. The activated SUMO proteins are transferred to form a thioester linkage with their conjugating (E2) enzyme, Ubc9. Finally, an isopeptide bond forms between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). Ulps/ SENPs can sever the linkage of SUMO proteins to their substrates; therefore, it is likely that SUMO modification is highly dynamic in vivo.

There are six members of the Ulp/SENP family in mammals and five in amphibians such as Xenopus laevis. We are systematically evaluating the physiological roles and regulation of these enzymes and are especially interested in the role of vertebrate Ulps/SENPs in the cell cycle and nuclear transport. In budding yeast, Ulps/SENPs are both cell cycle–regulated and required for mitotic events, particularly chromosome segregation. Similarly, yeast data suggest that Ulps/SENPs are controlled through localization to the nuclear pore complex (NPC) and that the SUMO pathway plays a significant role in nuclear trafficking.

We have shown that SENP6 (also called SUSP1) localizes within the nucleoplasm, where it plays a specialized role in dismantling highly conjugated SUMO-2 and SUMO-3 species. The function is similar to the chain-editing activity of SENP 6’s closest relative in budding yeast, Ulp2. We examined the role of SENP6 in chromosome segregation, a process that, in yeast, requires Ulp2p, and found that siRNA -mediated knockdown of SENP 6 in HeLa cells leads to chromosome misalignment. This phenotype is accompanied by defects in spindle assembly and mitotic progression. To understand the underlying molecular defect, we systematically evaluated the behavior of kinetochore-associated proteins, particularly those that are SUMOylated in other contexts. We found both microtubuledependent and microtubule-independent changes in kinetochore composition and are currently working to establish whether the alterations may be directly linked to the modification of individual kinetochore proteins.

We have found that two Ulp/SENP family members, SENP 3 and SENP 5, localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/Nucleophosmin. B23/ Nucleophosmin is an abundant shuttling phosphoprotein that plays important roles in ribosome biogenesis and has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/Nucleophosmin binds to SENP3 and SENP5 in Xenopus egg extracts and promotes the stability of SENP3 and SENP5 in mammalian tissue culture cells. After either co-depletion of SENP3 and SENP5 or depletion of B23/Nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulps/SENP s caused defects in ribosome biogenesis and export reminiscent of phenotypes observed in the absence of B23/Nucleophosmin.

Together, our results suggest that regulation of SUMO deconjugation may be a major facet of B23/Nucleophosmin function in vivo. In an effort to understand how SUMOylation contributes to ribosome biogenesis, we are currently examining SENP3 and SENP5 regulation and identifying ribosomal SUMOylation targets.

Dasso M. Emerging roles of the SUMO pathway in mitosis. CellDivision 2008;3:5-16.
Mukhopadhyay D, Ayaydin F, Kolli N, Tan S-H, Anan T, Kametaka A, Azuma Y, Wilkinson KD, Dasso M. SUSP1 antagonizes formation of highly SUMO-2/3 conjugated species. J Cell Biol 2006;174:939-949.
Mukhopadhyay D, Dasso M. Modification in reverse: the SUMO proteases. Trends Biochem Sci 2007;32:286-295.

Regulation of mitotic kinetochores by the Ran GTPase

Arnaoutov, Boyarchuk, Furuta, Ouspenskaia, Lukasiewicz; in collaboration with Yergey

The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly, and cell cycle control. The sole nucleotide exchange factor for Ran, RCC 1, binds to chromatin throughout the cell cycle. RanGAP 1, the GTP ase-activating protein for Ran, localizes to the cytosolic face of the nuclear pore complex (NPC) during interphase through association with RanBP2, a large nucleoporin. The interphase distribution of Ran regulators leads to a high concentration of Ran-GTP in nuclei and low Ran-GTP in cytosol. The major effectors for Ran are a family of Ran-GTP–binding proteins called karyopherins, which were discovered as nuclear transport receptors. Karyopherins transit the NPC in a Ran- and cargo-independent fashion. Karyopherins that mediate import are called importins, which bind to their cargo in the cytoplasm. Import complexes traverse the NPC and dissociate upon Ran-GTP-importin binding. Karyopherins that mediate export are called exportins, which bind to their cargo inside nuclei in complexes that contain Ran-GTP. After passage through the NPC, export complexes dissociate upon Ran-GTP hydrolysis. To date, two nuclear transport receptors have been shown to act as Ran effectors during mitosis, importin-beta and the exportin Crm1.

Our studies have focused on Ran functions at kinetochores, which are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis and serve as sites of spindle microtubule attachment. The kinetochore fibers (k-fibers) that link mammalian kinetochores to spindle poles contain microtubules that are directly attached to the kinetochores at their plus ends (kMT s) and microtubules that are not so attached. Kinetochore attachment is monitored through the spindle assembly checkpoint (SAC), which prevents mitotic exit by blocking anaphase-promoting complex/cyclosome (APC/C) activation until all chromosomes are attached and aligned onto the metaphase plate. The APC/C is a ubiquitin ligase that regulates the destruction of key mitotic regulatory proteins. Components of the SAC include Mad1, Mad2, Mps1, Bub1, Bub3, BubR1, and CENP-E. During mitosis, RanGAP1 and its binding partner RanBP2 associate with kinetochores in a microtubule-dependent fashion.

We showed that Crm1 localizes to kintochores and that inhibition of Crm1 ternary complex formation blocks recruitment of RanGAP1/RanBP2. Crm1 itself requires neither ternary complex assembly nor microtubules for kinetochore binding. We observed that, in the absence of Crm1 function, centromeres of mammalian tissue culture cells were under increased tension and that their kinetochores dramatically failed to maintain discrete attachments to single k-fibers. These defects caused increased chromosome mis-segregation. Our findings have several implications. First, they link RanGAP 1/RanBP2 to correct k-fiber assembly. Second, they suggest that Ran has a kinetochoreassociated effector pathway through Crm1. Third, they show that several karyopherins act as Ran effectors during mitosis; in principal, it is possible that other members of this family may also act during mitosis. The component(s) at kinetochores that is directly involved in Crm1 recruitment is a major focus of our current studies.

In addition, we have examined the function of SAC components within mitotic cells, particularly the Bub1 kinase’s role at the inner centromeres (IC) and its relationship with the chromosomal passenger complex (CPC). Protein complexes of the IC regulate sister chromatid cohesion and modulate MT attachment. The CPC consists of the Aurora B kinase, INCENP, Survivin, and Dasra/Borealin. Our findings suggest that Bub1 regulates localization of IC components through mechanisms that are both CPC-dependent and -independent. Remarkably, Bub1’s kinase activity is essential for IC assembly, in contrast to its roles at outer kinetochores.

Boyarchuk EY, Nikol’skii NN, Dasso M, Arnaoutov AM. Assembly of correct kinetochore architecture in Xenopus egg extract requires transition of spermDNA through interphase. Cell Tissue Biol 2007;1:80-88 (original Russian textpublished in Tsitologiya 49).
Boyarchuk Y, Salic A, Dasso M, Arnaoutov A. Bub1 is essential for assembly of the inner centromere. J Cell Biol 2007;176:919-928.
Dasso M. Ran at kinetochores. Biochem Soc Trans 2006;34:711-715.
Dasso M. The Ran GTPase: cellular roles and regulation. In: Hamm H, ed. Handbook of Cellular Signaling, Volume 3. Elsevier Science (USA), 2008, in press

Mitotic roles of nuclear pore complex proteins

Mishra, Ouspenskaia, Yoon; in collaboration with Yergey, Fontoura, Joseph

Vertebrate nuclear pore complexes (NPC) are large structures comprising about 30 distinct resident polypeptides (nucleoporins). During interphase, NPCs are conduits for transport of large substrates into the nucleus, allowing the passive diffusion of small molecules. Mutations in many nucleoporins are associated with human disease, including triple A syndrome, leukemias, and hematological malignancies as well as other cancers. During mitosis, metazoan NPCs disassemble into approximately a dozen subunits, at least two of which are targeted to mitotic kinetochores. The RanBP2 complex associates with kinetochores in a microtubule-dependent manner and consists of RanBP2 (a large nucleoporin also known as Nup358), SUMO-1–conjugated RanGAP1 (the activating protein for the Ran GTPase), and Ubc9 (the conjugating enzyme for the SUMO family of ubiquitin-like modifiers). The vertebrate Nup107–160 complex associates with kinetochores throughout mitosis in a microtubule-independent manner and includes Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Sec13, and Seh1.

Kinetochores are the primary site of attachment for chromosomes to the microtubules of the mitotic spindle and thus mediate the correct segregation of genetic material into daughter cells at anaphase. The SAC, which monitors kinetochore attachment, is a cell cycle–regulatory pathway that prevents the onset of anaphase until all chromosomes are properly attached to the mitotic spindle and aligned on the metaphase plate. Failure of the SAC is associated with chromosomal instability (CIN) leading to an abnormal chromosome number (aneuploidy). Such genetic alteration appears to be an important event in the origin of many human cancers. Our laboratory and others have shown that kinetochore-bound nucleoporins play essential roles in mitosis. We wish to understand these roles and to elucidate nucleoporin interactions with other kinetochore proteins. We use both in vivo (tissue culture cells) and in vitro (Xenopus laevis egg extracts) approaches to study these issues at a biochemical level, with an eventual goal of applying the knowledge gained to understanding the roles of NPC components in human disease.

During the past year, we demonstrated an interaction of the RanBP2 complex with the interphase cytoskeleton and found that a fraction of endogenous RanBP2 interacts with interphase microtubules through its N-terminal region (BPN). Overexpression of the BPN domain of RanBP2 caused dramatic increases in microtubule bundling and stability. Ectopic expression of BPN and full-length RanBP2 resulted in elevated levels of acetylated and detyrosinated alpha-tubulin, two markers for stable microtubules. Under the same circumstances, microtubules showed resistance to nocodazole, a depolymerizing agent. Depletion of RanBP2 disrupted polarized stabilization of microtubules during directed cell migration, showing that RanBP2 regulates interphase microtubules under physiological conditions. We are currently examining novel RanBP2 binding components and their role in regulation of RanBP2’s activity in microtubule dynamics during both interphase and mitosis.

Nup107–160 remains bound to the kinetochore throughout mitosis and shows enhanced accumulation on unattached kinetochores. We recently found that Nup107–160 interacts with an active form of the gamma-tubulin ring complex (gamma-TuRC), an essential and conserved microtubule nucleator. Like Nup107–160, gamma-TuRC localized to unattached mitotic kinetochores, prompting us to test whether Nup107–160 and gamma-TuRC might function in a coordinated manner to promote nucleation of microtubules near mitotic chromosomes and at kinetochores. Xenopus egg extracts lacking the Nup107–160 complex or gamma-TuRC failed to assemble spindles around sperm chromatin or DNA beads. Moreover, HeLa cells lacking Nup107–160 or gamma-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Our findings indicate that Nup107–160 promotes spindle assembly through the regulated nucleation of microtubules by gamma-TuRC at kinetochores and perhaps other sites within mitotic spindles. These observations suggest an important and novel relationship between the NPC and the microtubule cytoskeleton.

Our future studies will focus on several issues, including the components at kinetochores involved in recruitment of the Nup107–160 and RanBP2 complexes, the relationship between these complexes during mitosis, and how the complexes combine to regulate the attachment of microtubules to kinteochores and the spindle assembly checkpoint. We are also examining the mitotic localization and function of other NPC components.

Chakraborty P, Wang Y, Wei J-H, van Deursen J, Yu H, Malureanu L, Dasso M, Forbes DJ, Levy DE, Seemann J, Fontoura BMA. Nucleoporin levels regulate cellcycle progression and phase-specific gene expression. Dev Cell 2008;15:657-667.
Joseph J, Dasso M. The nucleoporin Nup358 associates with and regulates interphase microtubules. FEBS Lett 2008;582:190-196.

¹Graduate Partnerships Program

Collaborators

Beatriz M.A. Fontoura, PhD, University of Texas Southwestern Medical Center,Dallas, TX
Jomon Joseph, PhD, National Centre for Cell Science, Ganeshkhind, Pune, India
Keith D. Wilkinson, PhD, Emory University, Atlanta, GA
Alfred L. Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

For further information, contact mdasso@helix.nih.gov.