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MITOTIC REGULATION IN HIGHER EUKARYOTES
BY Ran AND SUMO-1
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Mary Dasso, PhD, Head,
Section on Cell Cycle Regulation Tadashi Anan, PhD, Visiting
Fellow Alexei Arnaoutov, PhD, Visiting
Fellow Ferhan Ayaydin, PhD, Visiting
Fellow Jomon Joseph, PhD, Visiting
Fellow Ai Kametaka, PhD, Visiting
Fellow Debaditya Mukhopadhyay, PhD, Visiting
Fellow Shyh Han Tan, PhD, Visiting
Fellow Chawon Yun, PhD, Visiting Fellow Yoshiaki Azuma, PhD, Research
Associate Byrn Booth Quimby, PhD, Postdoctoral
Fellow Yekaterina
Boyarchuk, BA, Predoctoral Fellow
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We
wish to understand the regulation of cell division in metazoan cells. Our
studies concentrate on two closely linked biochemical pathways that have been
implicated in both the regulation of mitosis and nuclear-cytoplasmic
trafficking: the SUMO conjugation pathway and the Ran GTPase pathway. SUMO
proteins are a family of ubiquitin-like proteins that become covalently
conjugated to cellular proteins in a manner similar to ubiquitin. In mammals,
three SUMO paralogs all use common enzymes for their conjugation. In recent
studies, we have investigated the specificity of paralog utilization, the
changing patterns of SUMO conjugation to individual substrates during the
cell cycle, and the behavior of enzymes that control SUMO conjugation and
deconjugation. The Ran GTPase is required for nucleocytoplasmic trafficking,
spindle assembly, nuclear assembly, and cell cycle control. We recently
documented that Ran functions through multiple mitotic effectors in mammalian
cells and plays critical roles at vertebrate kinetochores. We found that the
spindle checkpoint is responsive to Ran-GTP and that the Ran pathway is
closely regulated at the metaphase-anaphase transition. We have also shown
that SUMO-dependent targeting of Ran’s GTPase activating protein is
critical for spindle assembly and chromosome segregation. SUMO
family small ubiquitin-like modifiers in higher eukaryotes Anan, Ayaydin, Azuma, Kametaka,
Mukhopadhyay, Tan, Yun SUMO
proteins are a conserved family of ubiquitin-related proteins that become
conjugated to substrates in a manner similar to ubiquitin. Fission and
budding yeast each contain a single SUMO family protein. The proteins have
been implicated in the regulation of the cell cycle in both organisms. There
are three human SUMO paralogs: SUMO-1 is approximately 45 percent identical
to SUMO-2 and SUMO-3, which are 96 percent identical to each other. The
conjugation pathway for all paralogs is similar to the ubiquitin conjugation
pathway: SUMO proteins must be processed to yield a C-terminal di-glycine
motif. After processing, the first step in the SUMO conjugation pathway is
the ATP-dependent formation of a thioester bond between SUMO proteins and
their activating (E1) enzyme. The second step is the formation of a thioester
bond between SUMO proteins and their conjugating (E2) enzyme, Ubc9. In the
last step, an isopeptide bond is formed between SUMO proteins and substrates
through the cooperative action of Ubc9 and protein ligases (E3). An
important question about SUMO proteins concerns the roles of individual SUMO
paralogs within vertebrate cells. It is currently unclear whether SUMO-1, -2,
and -3 function in ways that are unique, redundant, or antagonistic.
Moreover, all three paralogs share common E1 and E2 enzymes while the
specificity of SUMO ligases and proteases is not well understood. It has been
difficult to address this question experimentally in the past because
superphysiological levels of individual SUMO proteins can cause a loss of
paralog specificity. Further, the dynamics and localizations of these
proteins can be only imprecisely estimated within fixed cells. To address the
dynamic properties of SUMO paralogs, we developed stable HeLa-derived cell
lines that express biofluorescent SUMO chimeras at levels comparable to those
of the endogenous proteins. Through live imaging and photobleaching studies,
we found that SUMO-1 differs from SUMO-2 and SUMO-3 in both its localization
and its dynamics throughout the cell cycle. In addition, we found significant
differences between SUMO-1 dynamics in different subnuclear compartments.
Together, our findings demonstrate that mammalian SUMO paralogs show discrete
temporal and spatial patterns of utilization throughout the cell cycle,
suggesting that the paralogs are functionally distinct and specifically
regulated in vivo. In
addition to experiments examining the role of SUMO-1 conjugation in the regulation
of RanGAP1 (see below), we sought to identify conjugation targets whose
modification is cell cycle–dependent. We found that Topoisomerase-II is
modified exclusively by SUMO-2/3 during mitosis in Xenopus egg
extracts; the modification is maximal in metaphase, followed by rapid
deconjugation during anaphase. The differential extraction properties of
modified and unmodified Topoisomerase-II suggest that SUMO-2/3 conjugation
may mobilize Topoisomerase-II from mitotic chromatin in a manner that is important
for chromosome segregation. Together, our findings indicate that SUMO-2/3
conjugation of Topoisomerase-II is important for the remodeling of mitotic
chromosomes at the metaphase-anaphase transition and that failure of such
remodeling could be expected to cause high levels of chromosome
mis-segregation in vivo. We identified the SUMO ligase that is
responsible for the mitotic conjugation of Topoisomerase-II and are currently
investigating how it is regulated by phosphorylation and association to chromatin. We
also systematically examined the properties and behavior of SUMO ligases
(PIAS/Siz family) and SUMO protease (SENPs). We find that PIAS family members
show similar patterns of localization within interphase nuclei yet also show
subtle differences in their localization on mitotic chromosomes. We
identified domains within one of the PIAS proteins (PIASy) that are
responsible for its targeting to interphase nuclei and to chromosomes.
Different SENP family members are localized to each of the following
organelles: nucleoli, nucleoplasm, and the nuclear envelope. We are currently
pursuing identification of the interactions responsible for such targeting
and are investigating the consequences of mis-targeting these enzymes. Ayaydin F, Dasso M. Distinct in vivo dynamics of vertebrate SUMO
paralogues. Mol Biol Cell 2004;15:5208-5218. Azuma Y, Arnaoutov A, Dasso M. SUMO-2 regulates Topoisomerase-II
in mitosis. J Cell Biol 2003;163:477-487. Azuma Y, Dasso M. A new clue at the nuclear pore: RanBP2 is an
E3 enzyme for SUMO1. Dev Cell 2002;2:130-131. Hang J, Dasso M. Association of the human SUMO-1 protease SENP2
with the nuclear pore. J Biol Chem 2002;277:19961-19966. Mitotic
roles of the Ran GTPase Arnaoutov, Boyarchuk,
Joseph, Quimby Ran is
a small GTPase required for nucleocytoplasmic trafficking, spindle assembly,
nuclear assembly, and cell cycle control. The nucleotide exchange factor for
Ran, RCC1, is a chromatin-associated protein. The GTPase-activating protein
for Ran, RanGAP1, is cytoplasmic during interphase. During mitosis, most of
the RanGAP1 is broadly distributed, although a significant fraction of
RanGAP1 becomes associated with kinetochores in a SUMO-1–dependent
fashion (see above). Ran-GTP nucleotide hydrolysis also requires a family of
Ran-GTP–binding proteins, which act as RanGAP1 accessory factors. The
family includes RanBP1 and RanBP2. It is widely held that the distribution of
Ran’s regulators plays a role is modulating local concentrations of
Ran-GTP within cells, spatially directing processes in which Ran has been
implicated. We have been examining the mechanisms through which important Ran
regulators are localized within mitotic metazoan cells and the functional
consequences to cells when such distribution patterns are disrupted. To
look at the mitotic fate of RCC1, we examined its chromosomal association in
cycling Xenopus egg extracts. Remarkably, the amount of
chromatin-associated RCC1 increased drastically at anaphase onset. To
determine the significance of this finding, we tested whether the Ran pathway
plays a role in mitotic progression or checkpoint control in Xenopus
egg extracts. Before each anaphase, chromosomes are aligned onto the
metaphase through attachment of spindle microtubules to kinetochores, which
are proteinaceous structures that assemble over the centromere of each
chromosome. The spindle assembly checkpoint is a cell cycle–regulatory
pathway that monitors spindle assembly in all eukaryotic cells and prevents
the onset of anaphase and the dissolution of sister chromatin cohesion in the
presence of unattached or inappropriately attached kinetochores. Remarkably,
a five- to seven-fold elevation of RCC1 concentration was sufficient to
abrogate spindle checkpoint arrest in extracts containing nuclei to which the
microtubule depolymerizing agent nocodazole had been added. While assembly of
centromeric structures occurred normally under these circumstances, we found
that many checkpoint components were mis-localized away from kinetochores
after RCC1 addition, indicating that increased RCC1 levels abolish checkpoint
arrest by altering interactions between kinetochores and checkpoint
regulators. Together with additional data, our observations suggest that the
spindle checkpoint is directly responsive to Ran-GTP levels. Notably, the
capacity of RCC1 to reverse spindle checkpoint arrest is specific, as
increased RCC1 does not compromise other modes of M phase arrest (e.g.,
cystostatic factor arrest). Our results suggest a model wherein complete
chromosome alignment on the metaphase plate triggers the increased binding of
RCC1 to chromosomes, resulting in the local elevation of Ran-GTP levels and
the ejection of the final population of kinetochore-associated checkpoint
components. In
parallel to our studies on RCC1, we have investigated the mitotic behavior
and function of RanGAP1. In metazoans, RanGAP1 is conjugated with SUMO-1.
Studies by our group and others have shown that SUMO-1 modification causes
RanGAP1 to associate during interphase with Ubc9 and RanBP2, a large nuclear
pore protein with multiple Ran-GTP–binding domains and a SUMO E3 ligase
domain. Through further investigation of the mitotic behavior and
interactions of RanGAP1, we found that RanGAP1 associates with kinetochores
in a SUMO-1–dependent manner. Notably, RanBP2 co-localized with RanGAP1
on spindles and kinetochores. More recently, we examined the structural
requirements for targeting RanGAP1 and RanBP2 as well as their function in
mitosis. We found that elimination of RanBP2 expression through RNA
interference (RNAi) displaced RanGAP1 from kinetochores, supporting the
notion that these proteins target to kinetochores as part of a single
complex. Both proteins were displaced after RNAi of integral kinetochore
components, suggesting that they require intact kinetochore structures to
localize appropriately. By contrast, peripheral kinetochore proteins were not
essential for correct targeting of either protein. Cells depleted of RanBP2
show abnormalities in both spindle
formation and mitotic progression, substantiating the importance of correct
targeting of the RanGAP1/RanBP2 complex during mitosis. Arnaoutov A, Dasso M. The Ran GTPase regulates kinetochore
function. Dev Cell 2003;5:99-111. Dasso M. The Ran GTPase: theme and variations. Curr Biol
2002;12:R502-R508. Joseph J, Liu S-T, Jablonski SA, Yen TJ, Dasso M. The
RanGAP1/RanBP2 complex is essential for microtubule-kinetochore interactions in
vivo. Curr Biol 2004;14:611-617. Joseph J, Tan SH, Karpova TS, McNally JG, Dasso M. SUMO-1 targets
RanGAP1 to the mitotic spindle. J Cell Biol 2002;156:595-602. Quimby BB, Dasso M. The small GTPase Ran: interpreting the
signs. Curr Opin Cell Biol 2003;15:338-344. COLLABORATOR Tim Yen, PhD, Fox Chase Cancer Center,
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