Fumiko Toyoshima

Nuclear export of cyclin B1 and its possible role in the DNA damage‐induced G2 checkpoint

M‐phase‐promoting factor (MPF), a complex of cdc2 and a B‐type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)‐dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES‐dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES‐disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES‐disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage‐induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage‐induced G2 checkpoint.

A novel SAPK/JNK kinase, MKK7, stimulated by TNFalpha and cellular stresses.

Stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinase (JNK), a member of the MAP kinase (MAPK) superfamily, is thought to play a key role in a variety of cellular responses. To date, SEK1/MKK4, one of the MAP kinase kinase (MAPKK) family of molecules, is the only SAPK/JNK kinase that has been cloned. Here we have cloned, identified and characterized a novel member of the mammalian MAPKKs, designated MKK7. MKK7 is most similar to the mediator of morphogenesis, hemipterous (hep), in Drosophila. Immunochemical studies have identified MKK7 as one of the major SAPK/JNK‐activating kinases in osmotically shocked cells. While SEK1/MKK4 can activate both the SAPK/JNK and p38 subgroups of the MAPK superfamily, MKK7 is specific for the SAPK/JNK subgroup. MKK7 is activated strongly by tumour necrosis factor α (TNFα) as well as by environmental stresses, whereas SEK1/MKK4 is not activated by TNFα. Column fractionation studies have shown that MKK7 is a major activator for SAPK/JNK in the TNFα‐stimulated pathway. Moreover, we have found that overexpression of MKK7 enhances transcription from an AP‐1‐dependent reporter construct. Thus, MKK7 is an evolutionarily conserved MAPKK isoform which is specific for SAPK/JNK, is involved in AP‐1‐dependent transcription and may be a crucial mediator of TNFα signalling.

Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner

The orientation of mitotic spindles is tightly regulated in polarized cells, but it has been unclear whether there is a mechanism regulating spindle orientation in nonpolarized cells. Here we show that integrin‐dependent cell adhesion to the substrate orients the mitotic spindle of nonpolarized cultured cells parallel to the substrate plane. The spindle is properly oriented in cells plated on fibronectin or collagen, but misoriented in cells on poly‐L‐lysine or treated with the RGD peptide or anti‐β1‐integrin antibody, indicating requirement of integrin‐mediated cell adhesion for this mechanism. Remarkably, this mechanism is independent of gravitation or cell–cell adhesion, but requires actin cytoskeleton and astral microtubules. Furthermore, myosin X and the microtubule plus‐end‐tracking protein EB1 are shown to play a role in this mechanism through remodeling of actin cytoskeleton and stabilization of astral microtubules, respectively. Our results thus uncover the existence of a mechanism that orients the spindle parallel to the cell–substrate adhesion plane, and identify crucial factors involved in this novel mechanism.

Polo-like kinase 1 facilitates chromosome alignment during prometaphase through BubR1.

Plk1, an evolutionarily conserved M phase kinase, associates with not only spindle poles but also kinetochores during prometaphase. However, the role of Plk1 at kinetochores has been poorly understood. Here we show that BubR1 mediates the action of Plk1 at kinetochores for proper chromosome alignment. Our results show that BubR1 colocalizes with Plk1 at kinetochores of unaligned chromosomes and physically interacts with Plk1 in prometaphase cells. Down-regulation of Plk1 by small interfering RNA abolished the mobility-shifted, hyperphosphorylated form of BubR1 in the prometaphase-arrested cells. In addition, BubR1 was phosphorylated by Plk1 in vitro at two Plk1 consensus sites in the kinase domain of BubR1. The add-back of either wild-type BubR1 or BubR1 2E, in which the two Plk1 phosphorylation sites were replaced by glutamic acids, but not that of BubR1 2A, an unphosphorylatable mutant, rescued the chromosome alignment defects in BubR1-deficient cells. Moreover, when both Plk1 and BubR1 were down-regulated, the add-back of BubR1 2E, but not that of wild-type BubR1, rescued the chromosome alignment defects. These results taken together suggest that Plk1 facilitates chromosome alignment during prometaphase through BubR1.

Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

ABSTRACT The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. βPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-βPix-actin pathway for this mechanism.

ABL1 regulates spindle orientation in adherent cells and mammalian skin.

Despite the growing evidence for the regulated spindle orientation in mammals, a systematic approach for identifying the responsible genes in mammalian cells has not been established. Here we perform a kinase-targeting RNAi screen in HeLa cells and identify ABL1 as a novel regulator of spindle orientation. Knockdown of ABL1 causes the cortical accumulation of Leu-Gly-Asn repeat-enriched-protein (LGN), an evolutionarily conserved regulator of spindle orientation. This results in the LGN-dependent spindle rotation and spindle misorientation. In vivo inactivation of ABL1 by a pharmacological inhibitor or by ablation of the abl1 gene causes spindle misorientation and LGN mislocalization in mouse epidermis. Furthermore, ABL1 directly phosphorylates NuMA, a binding partner of LGN, on tyrosine 1774. This phosphorylation maintains the cortical localization of NuMA during metaphase, and ensures the LGN/NuMA-dependent spindle orientation control. This study provides a novel approach to identify genes regulating spindle orientation in mammals and uncovers new signalling pathways for this mechanism.

Revolving movement of a dynamic cluster of actin filaments during mitosis

Arp2/3 actin filament nucleating complex drives circumnavigation of cortical actin clusters during mitosis.

Spindle orientation in animal cell mitosis : Roles of integrin in the control of spindle axis

The orientation of mitotic spindles, which determines the plane of cell division, is tightly regulated in polarized cells such as epithelial cells, but it has been unclear whether there is a mechanism regulating spindle orientation in non‐polarized cultured cells. In adherent cultured cells, spindles are positioned at the center of the cells and the axis of the spindle lies in the longest axis of the cell. Thus, cell geometry is thought to be one of cues for spindle orientation and positioning in cultured cells because this defines the center and the long axis of the cell. Recent work provides a new insight into the spindle orientation in cultured cells; spindles are aligned along the axis parallel to the cell‐substrate adhesion plane. Concomitantly, integrin‐mediated cell adhesion to the extracellular matrix (ECM), rather than gravitation, cell–cell adhesion or cell geometry, has shown to be essential for this mechanism of spindle orientation. Several independent lines of evidence confirm the involvement of cell‐ECM adhesion in spindle orientation in both cultured cells and in developing organisms. The important future challenge is to identify a molecular mechanism(s) that links integrin and spindles in the control of spindle axis. J. Cell. Physiol. 213: 407–411, 2007.

PCTK1 regulates integrin-dependent spindle orientation via protein kinase A regulatory subunit KAP0 and myosin X.

ABSTRACT Integrin-dependent cell-extracellular matrix (ECM) adhesion is a determinant of spindle orientation. However, the signaling pathways that couple integrins to spindle orientation remain elusive. Here, we show that PCTAIRE-1 kinase (PCTK1), a member of the cyclin-dependent kinases (CDKs) whose function is poorly characterized, plays an essential role in this process. PCTK1 regulates spindle orientation in a kinase-dependent manner. Phosphoproteomic analysis together with an RNA interference screen revealed that PCTK1 regulates spindle orientation through phosphorylation of Ser83 on KAP0, a regulatory subunit of protein kinase A (PKA). This phosphorylation is dispensable for KAP0 dimerization and for PKA binding but is necessary for its interaction with myosin X, a regulator of spindle orientation. KAP0 binds to the FERM domain of myosin X and enhances the association of myosin X-FERM with β1 integrin. This interaction between myosin X-FERM and β1 integrin appeared to be crucial for spindle orientation control. We propose that PCTK1-KAP0-myosin X-β1 integrin is a functional module providing a link between ECM and the actin cytoskeleton in the ECM-dependent control of spindle orientation.

Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis.

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.