A recent publication also brings Paskin in relation to the spindle checkpoint (Shaukat et al. 2012). The group of Stephen Gregory showed that a double knockdown of Paskin and Mad2 in Drosophila shows synthetic lethality. MAD2 is an important part of the spindle assembly checkpoint (SAC), which prevents cell cycle progression until all chromosomes are attached to the spindle apparatus correctly. Accurate duplication of the genetic information during cell division is important to suppress accumulation of mutations. In eukaryotes it is crucial not only that all the information is copied but also that all genomic material is distributed equally to the two daughter cells. Therefore, unattached kinetochores lead to the formation of the mitotic checkpoint complex (MCC). The main function of MCC is to inhibit the anaphase promoting complex, also called cyclosome (APC/C) (Sudakin et al. 2001). The APC/C acts as an E3 ubiquitin ligase. Several proteins to be marked for proteolytic degradation are recognized by APC/C (Pines 2011). Among them securin and cyclin B1 are the most important. Securin binds to separase preventing it from cleaving the SCC1 subunit of the cohesion ring. The cohesin ring is keeping the sister chromatides together preventing them from separation (Nasmyth and Haering 2009). Cyclin B1 is required for cyclin-dependent kinase 1 activity (Musacchio and Salmon 2007). The activation of APC/C induced by the lack of an unattached kinetochore signal therefore leads to the degradation of securin and cyclin B1. This allows the release of separase and the inactivation of CDK1 and ultimately the separation of the sister chromatides and mitotic exit (Lara-Gonzalez et al. 2012). As a part of the MCC complex, mitotic arrest deficiency 2 (MAD2) has a key role in regulating APC/C activity. MAD2 is crucial for the detection of the open kinetochore signal. MAD2 exists in two conformations. In the so called open conformation (o-MAD2), MAD2 is able to bind to MAD1. By changing to the closed conformation (c-MAD2), MAD1 is trapped within c- MAD2. The resulting MAD1-c-MAD2 complex is recruited to the open kinetochore (Shah et al. 2004). There, more MAD2 is recruited to the bound MAD1-c-MAD2 complex. Only the o-
MAD2 bound to MAD1-c-MAD2 is able to bind CDC20 thereby changing to c-MAD2 and trapping CDC20 (De Antoni et al. 2005). This CDC20-c-MAD2 complex recruits another subcomplex consisting of MAD3/BUBR1-BUB3 to form the MCC (Musacchio and Salmon 2007).
Fig. 4. Overview of the spindle assembly checkpoint. Unnattached kinetochores lead to the
formation of the mitotic checkpoint complex (MCC). The main role of the MCC is to inhibit the function of the anaphase promoting complex/cyclosome (APC/C). The inhibition of the ubiquitination of securin and cyclin B1 prevents kleisin cleavage and mitotic exit and therefore progression in the cell cycle (Lara-Gonzalez et al. 2012).
Impairment of the spindle assembly checkpoint among others is one cause for chromosomal instability (CIN). The abnormal number and structures of chromosomes resulting from CIN are associated with prognosis of a poor clinical outcome. CIN favours tumor evolution and therefore leads to increased metastasis and increased drug resistance. CIN can be induced by depleting MAD2 (Michel et al. 2001). The group of Stephen Gregory used a CIN model in
Drosophila in a synthetic lethality screen for phosphatases and kinases triggering apoptosis in
CIN cells (Shaukat et al. 2012). CIN was induced by knockdown of Mad2 by RNAi. Heterozygous Mad2 dsRNA expressing females were crossed with male homozygous for the
dsRNA allele of the investigated kinase. The resulting offspring were either CIN by Mad2 knockdown or not but in any case they expressed the kinase dsRNA. Thus a shift in the offspring ratio indicates increase of CIN specific lethality by the candidate gene. The
Drosophila kinome of 397 genes was screened. Interestingly, Paskin was among the candidate
kinases. The knockdown of Paskin showed a strong negative effect on viability in combination with a knockdown of Mad2. The ratio of Paskin dsRNA expressing offspring to Paskin and Mad2 dsRNA expressing offspring was 13.25. Furthermore, double knockdown of Paskin and Mad2 in wings resulted in tissue loss. Apparently, this tissue loss was caused by p53 dependent apoptosis. Additionally, immunostaining against γH2AX suggest increased DNA damage in Mad2, Paskin double knockdown wing discs compared to Paskin knockdown or Mad2, lacZ double knock downs. Importantly, Paskin knockdown alone showed no increase in anaphase defects suggesting no necessity for Paskin in chromosomal segregation. Additionally, similar amounts of cell death were seen in BubR1, Paskin double knockdown wing discs (Shaukat et al. 2012). These findings suggest that Paskin suppresses DNA damage caused cell death in a background with a weakened spindle assembly checkpoint in
Drososphila. Till now no research in a mammalian system on a putative role of PASKIN in
the context of the spindle assembly checkpoint has been published. Homozygous knockout of
Mad2 in mice results in death during early embryonic stages (Dobles et al. 2000). However,
heterozygous Mad2 mutant mice cells with reduced Mad2 levels show increased occurrence of premature sister chromatide separation and aneuploidy. Mice with a haplo-insufficiency for Mad2 are viable but develop lymphomas and lung tumours at a higher frequency than wildtype mice (Michel et al. 2001). Paskin knockout mice develop normally and no increase of tumour frequency has been reported (Katschinski et al. 2003). Since the experiments in
Drosophila show that only the double-insufficiency for Paskin and Mad2 show a strong
phenotype, it would be of great interest to investigate a crossing of the Paskin and the Mad2 knockout mice.