The analysis of Mps1p in budding yeast begs the question of whether the function of Mps1p in SPB duplication is conserved in other organisms, especially in vertebrates.
The possibility that Mps1 protein kinases are not general regulators for SPB- or centrosome duplication is provoked by the observation that the C. elegans genome lacks a clear Mps1p ortholog. Furthermore, fission yeast S. pombe Mph1p is clearly involved in the mitotic spindle checkpoint, but no function in SPB duplication could be observed (He et al., 1998).
During progression through the cell cycle, the centrosome needs to be duplicated once, and only once in animal cells. Normally, at the onset of mitosis, the duplicated centrosomes determine the bipolarity of the mitotic spindle (Raff, 2001).
However, deregulation of the centrosome duplication cycle leads to severe defects within a cell. Extra copies of centrosomes result in the formation of multipolar spindles, and a failure in centrosome duplication results in monopolar spindles. Both events will generally provoke abnormal chromosome segregation, leading to polyploid cells (Kramer and Ho, 2001; Nigg, 2002; Fisk et al., 2002). Importantly, many human tumor cells show aneuploidy and contain supernumerary centrosomes (Nigg, 2002; Lingle et al., 1998; Pihan et al., 1998; Weber et al., 1998). Therefore it is important to understand how centrosome duplication is regulated.
In animal cells, the centrosome was discovered and described almost simultaneously by Edouard van Beneden and Theodor Boveri, and it was depicted as a small body that seemed to control cell division (van Benenden, 1883; Boveri, 1887).
Electron microscopy revealed that the mammalian centrosome consists of two barrel-shaped centrioles that are embedded in a proteinaceous matrix of pericentriolar material (PCM) (Kellogg et al., 1994; Andersen, 1999b) (Fig. 7). Each centriole is composed of nine triplets of microtubules, thereby forming a 500nm long structure with a diameter of about 200nm. Importantly, the two centrioles can be distinguished from each other: the older of the two carries appendages that are close to its distal end. The appendages are implicated in anchoring microtubules (Bornens, 2002). The centrioles may contribute to PCM assembly and within the PCM many proteins such as γ-tubulin ring complexes are recruited that are essential for microtubule nucleation (Moritz and Agard, 2001) and for the formation of the mitotic spindle in prophase.
Figure 7: Structure of the centrosome in animal cells. The centrosome consists of two centrioles that are embedded in the PCM (pericentriolar material). Adopted from (Nigg, 2002)
At the end of mitosis each daughter cell inherits a single centrosome and by the start of the next mitosis each cell contains two centrosomes. Thus, during interphase, the centrosome has to be duplicated (Hinchcliffe and Sluder, 2001;
Stearns, 2001) (Fig. 8). In mammalian cells, this event starts in late G1/early S phase after loss of the orthogonal orientation of the two centrioles (Kuriyama and Borisy, 1981). The appearance of short daughter centrioles, so called pro-centrioles, at the proximal end of each parental centriole, indicates the beginning of centriole duplication at the beginning of S phase and during S phase (Kochanski and Borisy, 1990). Thus, centrosome duplication occurs by a semi-conservative mechanism (from the perspective of the entire centrosome). These procentrioles elongate during S and G2 phase, reaching mature length in mitosis and the following G1 (Kuriyama and Borisy, 1981; Lange et al., 2000). The completion of centrosome duplication takes place in G2 with the recruitment of several components of the PCM including γ-tubulin to increase microtubule-nucleation activity (Verde et al., 1992; Wolff et al., 1992; Lane and Nigg, 1996). Concomitantly, centrosome separation occurs with a pair of mother-daughter centrioles in each centrosome (Blangy et al., 1995; Walczak, 2000).
Figure 8: The centrosome duplication cycle.
Schematic illustration of the centrosome duplication cycle. The centrosome has to be duplicated once in every cell cycle to prevent chromosome missegregation or changes in ploidy. Modified and adopted from (Nigg, 2002).
On the molecular level, the coordination between DNA replication and centrosome duplication in S phase is mediated via the cyclin-dependent kinase 2 (Cdk2) (Hinchcliffe and Sluder, 2002). Cdk2 activity is required for both of these key S phase events. In addition, a common requirement for DNA replication and centrosome duplication is phosphorylation of the retinoblastoma gene product pRb and the subsequent release of the transcription factor E2F (Meraldi et al., 1999). E2F moves into the nucleus and switches on genes required for S phase, like cyclin A and E (Schulze et al., 1995; Ohtani et al., 1995). In some cell types like CHO or U2OS cells, the normal coordination between centrosome duplication and DNA replication can be disrupted by treating cells with hydroxyurea, which induces cell cycle arrest in S phase. If such cells are treated with hydroxyurea, DNA replication is blocked but centrosome duplication continues normally, leading to multiple copies (Balczon et al., 1995).
In Xenopus embryos, Cdk2 regulates centrosome duplication together with its binding partner cyclin E (Hinchcliffe et al., 1999; Lacey et al., 1999), whereas in mammalian somatic cells cyclin A has the predominant role in the Cdk2 complex (Meraldi et al., 1999; Balczon, 2001). Beside Cdk2, two other protein kinases have also been implicated in centrosome duplication. In the nematode Caenorhabditis elegans, the ZYG-1 kinase is required for centrosome duplication, and mutant
embryos arrested with single, unpaired centrioles and monopolar spindles (O'Connell et al., 2001). However, what kinase functionally resembles ZYG-1 in other organisms remains to be clarified. The other kinase involved in centrosome duplication is calcium-calmodulin kinase II (CaMKII) (Matsumoto and Maller, 2002). In Xenopus egg extracts, a sudden increase in calcium ions leads to activation of CaMKII, and this serves as the trigger for centrosome duplication. Inhibition of this kinase completely abolished centrosome duplication (Matsumoto and Maller, 2002). Thus, regulation of centrosome duplication by phosphorylation is an important mechanism, and the identification of substrates of the many kinases implicated in centrosome duplication is an important issue.
Little is known about the targets of Cdk2 in regulating centrosome duplication.
Putative substrates might include nucleophosmin (NPM/B23) (Okuda et al., 2000) and CP110 (Chen et al., 2002b), but the precise functions of these proteins within the centrosome duplication cycle have to be determined. In addition to nucleophosmin and CP110, a third potential substrate of Cdk2/cyclin E was recently described, namely mMps1, the mouse homolog of budding yeast S. cerevisiae Mps1p (Fisk and Winey, 2001), suggesting that mMps1 regulates centrosome duplication. It has been reported that mMps1 localizes to centrosomes throughout the cell cycle, both on the endogenous level and as a GFP-fusion protein (Fisk and Winey, 2001). Additionally, in mitosis it was localized to kinetochores. Furthermore, overexpression of mMps1 enhanced centrosome reduplication, whereas overexpression of a kinase-dead mutant, mMps1-KD, blocked centrosome reduplication in NIH3T3 cells that are arrested in S phase. In addition, Cdk2 activity was required for mMps1 driven centrosome reduplication, and Cdk2 could phosphorylate mMps1, at least in vitro, which was thought to stabilize mMps1, thus contributing to centrosome reduplication (Fisk and Winey, 2001).