Andrew M. Fry

Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A.

Centrosome duplication is a key requirement for bipolar spindle formation and correct segregation of chromosomes during cell division. In a manner highly reminiscent of DNA replication, the centrosome must be duplicated once, and only once, in each cell cycle. How centrosome duplication is regulated and coordinated with other cell-cycle functions remains poorly understood. Here, we have established a centrosome duplication assay using mammalian somatic cells. We show that centrosome duplication requires the activation of E2F transcription factors and Cdk2–cyclin A activity.

SUN1 Interacts with Nuclear Lamin A and Cytoplasmic Nesprins To Provide a Physical Connection between the Nuclear Lamina and the Cytoskeleton

ABSTRACT Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

Mitotic regulation by NIMA-related kinases

The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets.

APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box.

Nek2 is a NIMA‐related kinase implicated in regulating centrosome structure at the G2/M transition. Two splice variants have been identified that exhibit distinct patterns of expression during cell cycle progression and development. Here we show that Nek2A, but not Nek2B, is destroyed upon entry into mitosis coincident with cyclin A destruction and in the presence of an active spindle assembly checkpoint. Destruction of Nek2A is mediated by the proteasome and is dependent upon the APC/C–Cdc20 ubiquitin ligase. Nek2 activity is not required for APC/C activation. Nek2A destruction in early mitosis is regulated by a motif in its extreme C‐terminus which bears a striking resemblance to the extended destruction box (D‐box) of cyclin A. Complete stabilization of Nek2A requires deletion of this motif and mutation of a KEN‐box. Destruction of Nek2A is not inhibited by the cyclin B‐type D‐box, but the C‐terminal domain of Nek2A inhibits destruction of both cyclins A and B. We propose that recognition of substrates by the APC/C–Cdc20 in early mitosis depends upon possession of an extended D‐box motif.

Cell cycle regulation by the NEK family of protein kinases

Summary Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.

Early mitotic degradation of Nek2A depends on Cdc20- independent interaction with the APC/C

The temporal control of mitotic protein degradation remains incompletely understood. In particular, it is unclear why the mitotic checkpoint prevents the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of cyclin B and securin in early mitosis, but not cyclin A. Here, we show that another APC/C substrate, NIMA-related kinase 2A (Nek2A), is also destroyed in pro-metaphase in a checkpoint-independent manner and that this depends on an exposed carboxy-terminal methionine–arginine (MR) dipeptide tail. Truncation of the Nek2A C terminus delays its degradation until late mitosis, whereas Nek2A C-terminal peptides interfere with APC/C activity in an MR-dependent manner. Most importantly, we show that Nek2A binds directly to the APC/C, also in an MR-dependent manner, even in the absence of the adaptor protein Cdc20. As similar C-terminal dipeptide tails promote direct association of Cdc20, Cdh1 and Apc10–Doc1 with core APC/C subunits, we propose that this sequence also allows a substrate, Nek2A, to directly bind the APC/C. Thus, although Cdc20 is required for the degradation of Nek2A, it is not required for its recruitment and this renders its degradation insensitive to the mitotic checkpoint.

The Centrosomal Kinase Nek2 Displays Elevated Levels of Protein Expression in Human Breast Cancer

Aneuploidy and chromosome instability are common abnormalities in human cancer. Loss of control over mitotic progression, multipolar spindle formation, and cytokinesis defects are all likely to contribute to these phenotypes. Nek2 is a cell cycle-regulated protein kinase with maximal activity at the onset of mitosis that localizes to the centrosome. Functional studies have implicated Nek2 in regulation of centrosome separation and spindle formation. Here, we present the first study of the protein expression levels of the Nek2 kinase in human cancer cell lines and primary tumors. Nek2 protein is elevated 2- to 5-fold in cell lines derived from a range of human tumors including those of cervical, ovarian, breast, prostate, and leukemic origin. Most importantly, by immunohistochemistry, we find that Nek2 protein is significantly up-regulated in preinvasive in situ ductal carcinomas of the breast as well as in invasive breast carcinomas. Finally, by ectopic expression of Nek2A in immortalized HBL100 breast epithelial cells, we show that increased Nek2 protein leads to accumulation of multinucleated cells with supernumerary centrosomes. These data highlight the Nek2 kinase as novel potential target for chemotherapeutic intervention in breast cancer.

OFD1 Is a Centrosomal/Basal Body Protein Expressed during Mesenchymal-Epithelial Transition in Human Nephrogenesis

OFD1 is the gene responsible for the oral-facial-digital syndrome type 1, a cause of inherited cystic renal disease. The protein contains an N-terminal LisH motif, considered important in microtubule dynamics, and several putative coiled-coil domains. This study used a combination of microscopic, biochemical, and overexpression approaches to demonstrate that OFD1 protein is a core component of the human centrosome throughout the cell cycle. Using a series of GFP-OFD1 deletion constructs, it was determined that the N-terminus containing the LisH domain is not required for centrosomal localization; however, coiled-coil domains are critical, with at least two being necessary for centrosomal targeting. Importantly, most reported OFD1 mutations are predicted to cause protein truncation with loss of coiled-coil domains, presumably leading to loss of centrosomal localization. Kidney development constitutes a classic model of mesenchymal-epithelial transformation. By immunoprobing human metanephroi and kidney epithelial lines, it was found that, during acquisition of epithelial polarity, OFD1 became localized to the apical zone of nephron precursor cells and then to basal bodies at the origin of primary cilia in fully differentiated epithelia. These striking patterns of OFD1 localization within cells place the protein at key sites, where it may play roles not only in microtubule organization (centrosomal function) but also in mechanosensation of urine flow (a primary ciliary function).

Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction

During interphase, centrosomes are held together by a proteinaceous linker that connects the proximal ends of the mother and daughter centriole. This linker is disassembled at the onset of mitosis in a process known as centrosome disjunction, thereby facilitating centrosome separation and bipolar spindle formation. The NIMA (never in mitosis A)-related kinase Nek2A is implicated in disconnecting the centrosomes through disjoining the linker proteins C-Nap1 and rootletin. However, the mechanisms controlling centrosome disjunction remain poorly understood. Here, we report that two Hippo pathway components, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador (hSav1), directly interact with Nek2A and regulate its ability to localize to centrosomes, and phosphorylate C-Nap1 and rootletin. Furthermore, we demonstrate that the hSav1–Mst2–Nek2A centrosome disjunction pathway becomes essential for bipolar spindle formation on partial inhibition of the kinesin-5 Eg5. We propose that hSav1–Mst2–Nek2A and Eg5 have distinct, but complementary functions, in centrosome disjunction.

Polo-like Kinase-2 Is Required for Centriole Duplication in Mammalian Cells

Centriole duplication initiates at the G1-to-S transition in mammalian cells and is completed during the S and G2 phases. The localization of a number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling the centriole duplication cycle. Here we show that the human Polo-like kinase 2 (Plk2) is activated near the G1-to-S transition of the cell cycle. Endogenous and overexpressed HA-Plk2 localize with centrosomes, and this interaction is independent of Plk2 kinase activity. In contrast, the kinase activity of Plk2 is required for centriole duplication. Overexpression of a kinase-deficient mutant under S-phase arrest blocks centriole duplication. Downregulation of endogenous Plk2 with small hairpin RNAs interferes with the ability to reduplicate centrioles. Furthermore, centrioles failed to duplicate during the cell cycle of human fibroblasts and U2OS cells after overexpression of a Plk2 dominant-negative mutant. These results show that Plk2 is a physiological centrosomal protein and that its kinase activity is likely to be required for centriole duplication near the G1-to-S phase transition.