Chuanmao Zhang

Roles of Aurora Kinases in Mitosis and Tumorigenesis

Aurora kinases, which have been implicated in several vital events in mitosis, represent a protein kinase family highly conserved during evolution. The activity of Aurora kinases is delicately regulated, mainly by phosphorylation and degradation. Deregulation of Aurora kinase activity can result in mitotic abnormality and genetic instability, leading to defects in centrosome function, spindle assembly, chromosome alignment, and cytokinesis. Both the expression level and the kinase activity of Aurora kinases are found to be up-regulated in many human cancers, indicating that these kinases might serve as useful targets for the development of anticancer drugs. This review focuses on recent progress on the roles of Aurora kinases in mitosis and tumorigenesis. (Mol Cancer Res 2007;5(1):1–10)

Spatial and temporal coordination of mitosis by Ran GTPase

The small nuclear GTPase Ran controls the directionality of macromolecular transport between the nucleus and the cytoplasm. Ran also has important roles during mitosis, when the nucleus is dramatically reorganized to allow chromosome segregation. Ran directs the assembly of the mitotic spindle, nuclear-envelope dynamics and the timing of cell-cycle transitions. The mechanisms that underlie these functions provide insights into the spatial and temporal coordination of the changes that occur in intracellular organization during the cell-division cycle.

Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?

Ran is an abundant GTPase that is highly conserved in eukaryotic cells and has been implicated in many aspects of nuclear structure and function, especially determining the directionality of nucleocytoplasmic transport during interphase. However, cell-free systems have recently shown that Ran plays distinct roles in mitotic spindle assembly and nuclear envelope (NE) formation in vitro. During spindle assembly, Ran controls the formation of complexes with importins, the same effectors that control nucleocytoplasmic transport. Here, we review these advances and discuss a general model for Ran in the coordination of nuclear processes throughout the cell division cycle via common biochemical mechanisms.

Targeting of RCC1 to Chromosomes Is Required for Proper Mitotic Spindle Assembly in Human Cells

Ran GTPase is involved in several aspects of nuclear structure and function, including nucleocytoplasmic transport and nuclear envelope formation. Experiments using Xenopus egg extracts have shown that generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 also plays roles in mitotic spindle assembly. Here, we have examined the localization and function of RCC1 in mitotic human cells. We show that RCC1, either the endogenous protein or that expressed as a fusion with green fluorescent protein (GFP), is localized predominantly to chromosomes in mitotic cells. This localization requires an N-terminal lysine-rich region that also contains a nuclear localization signal and is enhanced by interaction with Ran. Either mislocalization of GFP-RCC1 by removal of the N-terminal region or the expression of dominant Ran mutants that perturb the GTP/GDP cycle causes defects in mitotic spindle morphology, including misalignment of chromosomes and abnormal numbers of spindle poles. These results indicate that the generation of Ran-GTP in the vicinity of chromosomes by RCC1 is important for the fidelity of mitotic spindle assembly in human cells. Defects in this system may result in abnormal chromosome segregation and genomic instability, which are characteristic of many cancer cells.

Role of Importin-β in the Control of Nuclear Envelope Assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.

Roles of Ran–GTP and Ran–GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system

The molecular mechanism of nuclear envelope (NE) assembly is poorly understood, but in a cell-free system made from Xenopus eggs NE assembly is controlled by the small GTPase Ran [1,2]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [1]. Both generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 and GTP hydrolysis by Ran are required for NE assembly, although the roles of the GDP- and GTP-bound forms of Ran in the recruitment of precursor vesicles and their fusion have been unclear. We now show that beads coated with either Ran-GDP or Ran-GTP assemble functional nuclear envelopes in a cell-free system derived from mitotic human cells, forming pseudo-nuclei that actively transport proteins across the NE. Both RCC1 and the GTPase-activating protein RanGAP1 are recruited to the beads, allowing interconversion between Ran-GDP and Ran-GTP. However, addition of antibodies to RCC1 and RanGAP1 shows that Ran-GDP must be converted to Ran-GTP by RCC1 before precursor vesicles are recruited, whereas GTP hydrolysis by Ran stimulated by RanGAP1 promotes vesicle recruitment and is necessary for vesicle fusion to form an intact envelope. Thus, the GTP-GDP cycle of Ran controls both the recruitment of vesicles and their fusion to form NEs.

Lamin B receptor plays a role in stimulating nuclear envelope production and targeting membrane vesicles to chromatin during nuclear envelope assembly through direct interaction with importin β

Lamin B receptor (LBR), a chromatin and lamin binding protein in the inner nuclear membrane, has been proposed to play a vital role in nuclear envelope (NE) assembly. But the specific role for LBR in NE assembly remains unknown. In the present study, we show that overexpression of LBR causes membrane overproduction, inducing NE invagination and membrane stack formation, and that these processes require the transmembrane domain of LBR. Biochemical analysis shows that the N-terminal domain of LBR directly interacts with importin β in a Ran sensitive and importin α independent manner. Using an in vitro NE assembly assay, we also demonstrate that blocking full length LBR binding sites on importin β, by the addition of the LBR N-terminal domain inhibits the recruitment of LBR-containing vesicles to importin β- or Ran-coated beads to form NE structure. Our results suggest that LBR is recruited to chromatin through direct interaction with importin β to contribute to the fusion of membrane vesicles and formation of the NE.

Sequential phosphorylation of Nedd1 by Cdk1 and Plk1 is required for targeting of the γTuRC to the centrosome

Nedd1 is a new member of the γ-tubulin ring complex (γTuRC) and targets the γTuRC to the centrosomes for microtubule nucleation and spindle assembly in mitosis. Although its role is known, its functional regulation mechanism remains unclear. Here we report that the function of Nedd1 is regulated by Cdk1 and Plk1. During mitosis, Nedd1 is firstly phosphorylated at T550 by Cdk1, which creates a binding site for the polo-box domain of Plk1. Then, Nedd1 is further phosphorylated by Plk1 at four sites: T382, S397, S637 and S426. The sequential phosphorylation of Nedd1 by Cdk1 and Plk1 promotes its interaction with γ-tubulin for targeting the γTuRC to the centrosome and is important for spindle formation. Knockdown of Plk1 by RNAi decreases Nedd1 phosphorylation and attenuates Nedd1 accumulation at the spindle pole and subsequent γ-tubulin recruitment at the spindle pole for microtubule nucleation. Taken together, we propose that the sequential phosphorylation of Nedd1 by Cdk1 and Plk1 plays a pivotal role in targeting γTuRC to the centrosome by promoting the interaction of Nedd1 with the γTuRC component γ-tubulin, during mitosis.

PCM1 recruits Plk1 to the pericentriolar matrix to promote primary cilia disassembly before mitotic entry

Summary Primary cilia, which emanate from the cell surface, exhibit assembly and disassembly dynamics along the progression of the cell cycle. However, the mechanism that links ciliary dynamics and cell cycle regulation remains elusive. In the present study, we report that Polo-like kinase 1 (Plk1), one of the key cell cycle regulators, which regulate centrosome maturation, bipolar spindle assembly and cytokinesis, acts as a pivotal player that connects ciliary dynamics and cell cycle regulation. We found that the kinase activity of centrosome enriched Plk1 is required for primary cilia disassembly before mitotic entry, wherein Plk1 interacts with and activates histone deacetylase 6 (HDAC6) to promote ciliary deacetylation and resorption. Furthermore, we showed that pericentriolar material 1 (PCM1) acts upstream of Plk1 and recruits the kinase to pericentriolar matrix (PCM) in a dynein-dynactin complex-dependent manner. This process coincides with the primary cilia disassembly dynamics at the onset of mitosis, as depletion of PCM1 by shRNA dramatically disrupted the pericentriolar accumulation of Plk1. Notably, the interaction between PCM1 and Plk1 is phosphorylation dependent, and CDK1 functions as the priming kinase to facilitate the interaction. Our data suggest a mechanism whereby the recruitment of Plk1 to pericentriolar matrix by PCM1 plays a pivotal role in the regulation of primary cilia disassembly before mitotic entry. Thus, the regulation of ciliary dynamics and cell proliferation share some common regulators.

Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment.

Transforming acidic coiled-coil-containing protein 3 (TACC3) has been implicated in mitotic spindle assembly, although the mechanisms involved are largely unknown. Here we identify that clathrin heavy chain (CHC) binds specifically to phosphorylated TACC3 and recruits it to spindle poles for proper spindle assembly and chromosome alignment. Phosphorylation of Xenopus TACC3 at serine 620 (S620) and S626, but not S33, is required for its binding with CHC. Knockdown of CHC by RNA interference (RNAi) abolishes the targeting of TACC3 to spindle poles and results in abnormal spindle assembly and chromosome misalignment, similar to the defects caused by TACC3 knockdown. Furthermore, the binding of CHC with phosphorylated TACC3 is inhibited by importin β and this inhibition is reversed by the presence of the GTP-binding nuclear protein Ran in the GTP-bound state. Together, these results indicate that the recruitment of phosphorylated TACC3 to spindle poles by CHC ensures proper spindle assembly and chromosome alignment, and is regulated by Ran.