Many hormones, growth factors, and cytokines regulate proliferation of their target cells. Perhaps the most universal signaling cascades required for proliferative responses are those initiated by transient rises in intracellular calcium (Ca(2+)). The major intracellular receptor for Ca(2+) is calmodulin (CaM). CaM is a small protein that contains four EF-hand Ca(2+) binding sites and is highly conserved among eukaryotes. In all organisms in which the CaM gene has been deleted, it is essential. Although Ca(2+)/CaM is required for proliferation in both unicellular and multicellular eukaryotes, the essential targets of Ca(2+)/CaM-dependent pathways required for cell proliferation remain elusive. Potential Ca(2+)/CaM-dependent targets include the serine/threonine phosphatase calcineurin and the family of multifunctional Ca(2+)/CaM-dependent protein kinases. Whereas these enzymes are essential in Aspergillus nidulans, they are not required under normal growth conditions in yeast. However, in mammalian cells, studies demonstrate that both types of enzymes contribute to the regulation of cell cycle progression. Unfortunately, the mechanism by which Ca(2+)/CaM and its downstream targets, particularly calcineurin and the Ca(2+)/CaM-dependent protein kinases, regulate key cell cycle-regulatory proteins, remains enigmatic. By understanding how Ca(2+)/CaM regulates cell cycle progression in normal mammalian cells, we may gain insight into how hormones control cell division and how cancer cells subvert the need for Ca(2+) and its downstream targets to proliferate.

Regulation of cell cycle progression by calcium/calmodulin-dependent pathways

Nek2, a mammalian protein kinase of unknown function, is closely related to the mitotic regulator NIMA of Aspergillus nidulans. Here we show by both immunofluorescence microscopy and biochemical fractionation that human Nek2 localizes to the centrosome. Centrosome association occurs throughout the cell cycle, including all stages of mitosis, and is independent of microtubules. Overexpression of active Nek2 induces a striking splitting of centrosomes, whereas prolonged expression of either active or inactive Nek2 leads to dispersal of centrosomal material and loss of a focused microtubule-nucleating activity. Surprisingly, this does not prevent entry into mitosis, as judged by the accumulation of mitotically arrested cells induced by co-expression of a non-destructible B-type cyclin. These results bear on the dynamic function of centrosomes at the onset of mitosis. Moreover, they indicate that one function of mammalian Nek2 relates to the centrosome cycle and thus provide a new perspective on the role of NIMA-related kinases.

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A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators.

Recent studies have implicated centrosome amplification in the origin of chromosomal instability during tumor development. Theodor Boveri first suggested this notion nearly a century ago (Boveri, 1914). While the role of centrosome amplification in the origin of malignant tumors remains a controversial issue, several emerging lines of evidence suggest that cell cycle pathways converge on the centrosome and implicate this organelle in the control of cell cycle progression in addition to its function as a microtubule organizing center. Here we review the relationship between cell cycle regulation and centrosome amplification in the development of cancer. The centrosome influences cell structure through the nucleation and organization of cytoplasmic microtubules (Kirschner and Mitchison, 1986). Interphase cells contain a single centrosome that is typically located near the nucleus and contains of a pair of centrioles that anchor the recruitment of pericentriolar material including the microtubule nucleating protein g-tubulin (Bobinnec et al., 1998). Centrioles are small organelles (*200 nm diameter and 400 nm in length) consisting of a cylindrical array of nine triplet microtubules (Dutcher, 2001a,b). The centrosome is duplicated once, and only once, during a normal cell cycle to give rise to two centrosomes that function as the spindle poles of the dividing cell (Kellogg, 1989). This process is most clearly illustrated by duplication of the centrioles themselves. In early G1 phase of the cell cycle the two centrioles are typically oriented in a characteristic orthogonal arrangement. As cells pass the G1 restriction point and commit to DNA replication and subsequent cell division, the two centrioles separate a short distance from one another and nascent procentrioles form at the proximal end and orthogonal to each pre-existing centriole (Adams and Kilmartin, 2000; Wheatley, 1982). During G2/M phase centrosome duplication is completed and each new centrosome (i.e. mitotic spindle pole) contains one old and one new centriole. The presence of only two centrosomes in the cell as it enters mitosis ensures the equal segregation of sister chromatids to each daughter cell. Mitotic spindle poles also play a role in determining the position and orientation of the cleavage furrow and in exit from cytokinesis (Khodjakov and Rieder, 2001; Piel et al., 2001).

Centrosome amplification and the development of cancer.

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly.

Intracellular targeting may enable protein kinases with broad substrate-specificities, such as multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) to achieve a selectivity of action in vivo. We have examined the intracellular targeting of three delta-CaM kinase isoforms. The delta B-CaM kinase isoform is targeted to the nucleus in transfected cells while the delta A- and delta C-CaM kinase isoforms are cytosolic/cytoskeletal. A chimeric construct of alpha-CaM kinase containing the delta B-CaM kinase variable domain is rerouted to the nucleus while the native alpha-CaM kinase and chimeras of alpha-CaM kinase which contain the delta A- or delta C-CaM kinase variable domains are retained in the cytoplasm. Using site-directed mutagenesis, we have defined a nuclear localization signal (NLS) within an 11-amino acid sequence, likely inserted by alternative splicing, in the variable domain of delta B-CaM kinase. Isoform-specific nuclear targeting of CaM kinase is probably a key mechanism in the selective regulation of nuclear functions by CaM kinase. CaM kinase is a multimer that can be composed of several isoforms. We find that when cells express two different isoforms of CaM kinase, cellular targeting is determined by the ratio of the isoforms. When an excess of the cytoplasmic isoform of CaM kinase is coexpressed along with the nuclear isoform, both isoforms are localized in the cytoplasm. Conversely an excess of the nuclear isoform can reroute the cytoplasmic isoform to the nucleus. The nuclear isoform likely coassembles with the cytosolic isoform, to form a heteromultimeric holoenzyme which is transported into the nucleus. These experiments demonstrate isoform-specific targeting of CaM kinase and indicate that such targeting can be modified by the expression of multiple isoforms of the enzyme.

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Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus.

Indirect immunofluorescence was used to determine the distribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in rat embryo fibroblast 3Y1 cells, rat C6 glioma cells, and human epidermoid carcinoma KB cells. During interphase at growing phase, CaM kinase II was localized diffusely in the cytoplasm and in the nucleus. In the nucleus, the enzyme was localized within the whole nuclear matrix in which the enzyme was specially concentrated in nucleoli. During mitosis, CaM kinase II was found to be a dynamic component of the mitotic apparatus, particularly present at microtubule-organizing centers. In metaphase and anaphase, CaM kinase II was observed at centrosomes and between the spindle poles. During telophase, CaM kinase II was condensed as a bright fluorescent dot at the midzone of the intercellular bridge between two daughter cells, while tubulin was found at each side of the midbody. Colchicine, a microtubule inhibitor, disorganized the tubulin- and CaM kinase II specific fluorescent structure of mitotic 3Y1 cells. In cold-treated cells, CaM kinase II was localized predominantly at centrosomes. The localization of CaM kinase II in the cell nucleus and the mitotic apparatus suggests that the enzyme may play a role in the cell cycle progression of mammalian cells.

https://www.pnas.org/content/pnas/87/14/5341.full.pdf

Eishichi Miyamoto

Papers

Ca2+/calmodulin-dependent protein kinase II: localization in the interphase nucleus and the mitotic apparatus of mammalian cells.