Introduction
For accurate chromosome segregation, attachment of sister kinetochores on a replicated chromosome to microtubules from opposite spindle poles (bi‐orientation) must be achieved (
Tanaka et al, 2005;
Tanaka, 2008;
Walczak et al, 2010). Thus far, a number of molecules have been identified that are essential for accurate chromosome segregation, including molecules directly involved in kinetochore–microtubule attachment (
Cheeseman et al, 2006;
Cheeseman and Desai, 2008;
Tanaka and Desai, 2008) and the correction of erroneous attachments (
Ruchaud et al, 2007), as well as components of the spindle assembly checkpoint (SAC) that prevents anaphase onset until proper kinetochore–microtubule attachment to all chromosomes has been achieved (
Musacchio and Salmon, 2007). Many of these molecules are conserved from yeast to human, but some are found specifically in mammalian cells where they are suggested to be involved in the precise regulation of mammalian kinetochore–microtubule attachment. Identification of such molecules will not only allow us to further understand the control of chromosome segregation in human cells, but could also have clinical significance, as dysregulation of chromosome segregation may lead to oncogenic transformation through the induction of chromosomal instability (
Weaver and Cleveland, 2005;
Ganem et al, 2007;
Ricke et al, 2008;
Tanaka and Hirota, 2009).
Here, we report a novel regulator for accurate chromosome segregation,
chromosome
alignment‐
maintaining
phosphoprotein (CAMP). We identified CAMP as a MAD2L2‐interacting protein. MAD2L2, together with MAD2L1, is an orthologue of yeast MAD2 that has a central function in the SAC (
Cahill et al, 1999). While MAD2L1 is the canonical MAD2, MAD2L2 shares homology with yeast REV7, a component of polymerase ζ (pol ζ), which is involved in translesion synthesis (
Gan et al, 2008). In contrast to MAD2L1, which inhibits activation of the anaphase‐promoting complex/cyclosome (APC/C) through its interaction with CDC20 (
Musacchio and Salmon, 2007), MAD2L2 inhibits APC/C through binding with another APC/C cofactor, CDH1 (
Chen and Fang, 2001;
Pfleger et al, 2001). APC/C
Cdh1 is involved in the degradation of Cyclin B1, CDC20, and Plk1, all of which are required for mitotic progression (
Baker et al, 2007). Accordingly, MAD2L2‐depleted cells show defective mitotic entry (
Iwai et al, 2007). In other reports, MAD2L2‐depleted cells exhibited a defective DNA damage response, suggesting that MAD2L2 is a functional homologue of yeast REV7 (
Okada et al, 2005;
Cheung et al, 2006). These data suggest that MAD2L2 has at least two functions, mitotic control and DNA damage response. However, its potential role in chromosome segregation has not been explored yet.
CAMP is a zinc‐finger protein conserved in vertebrates and contains several characteristic repeat motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and is phosphorylated during mitosis. Interestingly, it is involved in kinetochore–microtubule attachment in a way that maintains chromosome alignment on the metaphase plate. Our domain analyses identified a novel functional domain with the unique FPE repeat motifs responsible for the CAMP function on proper chromosome segregation, which appears to be regulated by the mitotic phosphorylation.
Discussion
In this paper, we report the identification of a novel protein CAMP, a phosphoprotein containing unique repeat motifs, which appears to be involved in kinetochore–microtubule attachment. In addition to the canonical C2H2 zinc‐finger domains, CAMP contains several characteristic repeat motifs (
Supplementary Figure S1C;
Figure 8). Among them, the SPE motifs contain serine residues that are phosphorylated during mitosis. The WK motifs, which partially overlap with the SPE motifs, mediate the interaction between CAMP and MAD2L2 (
Supplementary Figure S1E). Spindle localization of MAD2L2 was abrogated by CAMP depletion (
Supplementary Figure S2A), although the role of MAD2L2 on the spindle has not been clarified yet (
Medendorp et al, 2009). We did not observe a delay in mitotic entry in CAMP‐depleted cells (data not shown) as was reported for MAD2L2‐depleted cells (
Iwai et al, 2007). We also did not observe chromosome misalignment in MAD2L2‐depleted cells (
Supplementary Figure S2A), and the WK region was found to be dispensable for chromosome alignment (
Figure 5A and B). Thus, the functional relationship between CAMP and MAD2L2 awaits further investigation. The region containing the FPE motifs is involved in spindle/kinetochore localization, but not in chromosome localization (
Figures 1C–E and
7C), and is essential for chromosome alignment (
Figure 5A and B). The C‐terminal region containing the zinc‐finger domains is involved in the localization of CAMP to both chromosomes and the spindle (
Figure 1C and D), and has an inhibitory effect on chromosome alignment (
Figure 6D). Concerning the localization of CAMP throughout chromosome length, we examined the localizations of the chromokinesin Kid, HP1α, condensin I and II, all of which are found on chromosomes, as well as the phosphorylation status of serine 10 or threonine 3 of histone H3 (
Dai et al, 2005;
Supplementary Figure S7B–E). We did not observe any difference in CAMP‐depleted cells compared with mock‐treated cells, leaving the function of chromosome‐localized CAMP an unanswered question. CAMP is a unique protein, which does not form an ordered structure other than zinc‐finger domains, as predicted by the PrDOS server (
http://prdos.hgc.jp/cgi-bin/top.cgi;
Ishida and Kinoshita, 2007). Disordered regions are frequently involved in interaction with other proteins and often contain phosphorylation sites (
Iakoucheva et al, 2004;
Dyson and Wright, 2005). CAMP may have multiple functions mediated by the disordered regions containing characteristic repeat motifs. Investigation of the structures and functions of these motifs and search for similar motifs in other proteins are important future directions.
Our data suggest that kinetochore–microtubule attachment in CAMP‐depleted cells was too weak to resist tension applied between sister kinetochores. How does CAMP regulate kinetochore–microtubule attachment? The FPE region, which can localize spindle/kinetochores (
Figures 1C–E and
7C), was found to be essential for the function (
Figure 5A and B). As CAMP did not bind to microtubules in our
in vitro assay (SC, KM, and KT unpublished observations), we consider it unlikely that CAMP is involved directly in kinetochore–microtubule interaction at kinetochores. Therefore, one plausible possibility is that CAMP affects the localization/function of other molecules involved in kinetochore–microtubule attachment on spindle/kinetochores. We found that CENP‐E and CENP‐F decreased on kinetochores in CAMP‐depleted cells (
Figure 7A and B). CENP‐E and CENP‐F reside at the kinetochore–microtubule interface and are involved in stabilizing the binding of microtubules to kinetochores (
Yao et al, 2000;
McEwen et al, 2001;
Putkey et al, 2002;
Bomont et al, 2005;
Feng et al, 2006;
Vergnolle and Taylor, 2007). CENP‐E and CENP‐F are closely related proteins, as they are known to interact each other and their kinetochore localization is interdependent (
Chan et al, 1998;
Johnson et al, 2004). Moreover, both are farnesylated, which is crucial for their mitotic functions (
Ashar et al, 2000;
Hussein and Taylor, 2002;
Schafer‐Hales et al, 2007). Colocalization of CAMP with CENP‐E/CENP‐F on kinetochores (
Figure 7C) and the strong correlation between CAMP functionality and CENP‐E/CENP‐F levels on kinetochores (
Figures 6D and
7F;
Supplementary Figure S9) supports the notion that CAMP regulates targeting CENP‐E/CENP‐F to kinetochores. Kinetochore localization of CENP‐E/CENP‐F is regulated directly by interaction with other kinetochore components (
Chan et al, 1998;
Johnson et al, 2004;
Liu et al, 2007), or indirectly by posttranslational modifications (
Schafer‐Hales et al, 2007;
Zhang et al, 2008). As we found no evidence for physical interaction between CAMP and CENP‐E/CENP‐F (unpublished data), CAMP may regulate localization of CENP‐E/CENP‐F indirectly through one of these mechanisms. Lack of additive effects on chromosome misalignment when CAMP and CENP‐E/CENP‐F were simultaneously depleted (
Figure 7D) suggests that CENP‐E and CENP‐F are downstream of CAMP in mitotic function. When we quantified the number of misaligned chromosomes per cell, however, more chromosomes were misaligned in CAMP‐depleted cells compared to CENP‐E/CENP‐F‐depleted cells (
Supplementary Figure S8B and C), suggesting that targeting CENP‐E/CENP‐F to kinetochores is not the only mechanism for chromosome alignment by CAMP.
Another possibility is that CAMP may function as a (indirect) microtubule‐associated protein to regulate microtubule dynamics necessary for proper chromosome segregation (
Wong and Fang, 2006;
Yokoyama et al, 2009). In CAMP‐depleted cells, most of the chromosomes aligned on the metaphase plate, but the cells could not maintain chromosome alignment. Similar phenotype was also reported in cells in which RAMA1/Ska3 is depleted (
Raaijmakers et al, 2009;
Theis et al, 2009). RAMA1/Ska3 is a component of the Ska complex, which localizes to the spindle and kinetochores (
Hanisch et al, 2006;
Daum et al, 2009;
Gaitanos et al, 2009;
Raaijmakers et al, 2009;
Theis et al, 2009;
Welburn et al, 2009), but in this case it binds directly to microtubules (
Welburn et al, 2009). The Ska complex is involved in stable kinetochore–microtubule interactions (
Gaitanos et al, 2009;
Raaijmakers et al, 2009;
Theis et al, 2009) and may couple kinetochores with microtubule depolymerization (
Welburn et al, 2009), as shown previously for the Dam1 complex in budding yeast (
Asbury et al, 2006;
Westermann et al, 2006;
Tanaka et al, 2007). Multipolar spindles in CAMP‐depleted cells (
Supplementary Figure S3A) probably reflect defective kinetochore–microtubule attachment or defective microtubule organization, resulting indirectly in altered spindle structure, as was observed with Ska1 or RAMA1 depletion (
Welburn et al, 2009).
CAMP is phosphorylated at serine residues in multiple SPE/D sequences, which reduces its electrophoretic mobility (
Figure 6B). We found that mitotic phosphorylation of CAMP is dependent on CDK1 (
Figure 6C). It is possible that CDK1, a proline‐directed kinase, phosphorylates the SPE/D sequences, although direct phosphorylation of CAMP by CDK1 needs to be proven. Mitotic phosphorylation of CAMP was essential for proper chromosome alignment (
Figure 6D). Interestingly, expression of a CAMP mutant with a non‐phosphorylatable FPE region did not rescue chromosome misalignment, but phosphorylation became dispensable when CAMP lacked the C‐terminal region (
Figure 6D). These data suggest that the C‐terminal region inhibits the function of the FPE region, and that this inhibition is relieved by serine phosphorylation of the FPE region (
Figure 8). One possibility is that the C‐terminal region inhibits binding of molecules required for stable kinetochore–microtubule attachment to the FPE region. Alternatively, the C‐terminal region binds and thereby masks the FPE region, and this intra‐molecular association is counteracted by phosphorylation. As CAMP is conserved only among vertebrates, it is likely to be involved in the fine regulation of chromosome segregation in higher eukaryotes, and dysregulation of its function could be related to oncogenesis.
Acknowledgements
We thank J‐M Peters for EGFP‐CENP‐A/EGFP‐α‐tubulin cell; A Harata, T Aizawa, S Arai, and S Shimanuki for technical assistance; and TU Tanaka, M Satake, and T Shiraki for helpful discussions. This work was supported by Special Coordination Funds for Promoting Science and Technology from the Japan Science and Technology Agency; a Grant‐in‐Aid for Scientific Research from the Japanese Society of Promotion of Science; a Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a grant from the Tohoku University ‘Evolution’ program; and grants from the Naito Foundation, the NOVARTIS Foundation, and the Takeda Science Foundation.
Author contributions: SK carried out the far western analysis, KSKU contributed to the live‐cell imaging, SC, SS, KW, and KM were involved in the biochemical experiments, and AY contributed to the MS work. GI performed all other experiments. TH and KT supervised research and wrote the paper.