Department of Molecular Genetics

Fig. 1: The functions of the Lats and GAK complexes correlate closely.

We are studying the eukaryotic cell cycle to understand the mechanism that is responsible for the chromosome instability in cancer cells. Chromosome instability is observed in cancer cells, but not in normal cells. Indeed, many human cancer cells exhibit mitotic defects (such as centrosome aberrations, abnormal spindle formation, and chromosome missegregation), and the resulting chromosome instability has been shown to be a major cause of malignant tumor progression. We are focusing on functional analyses of the Ser/Thr kinases Lats (large tumor suppressor) and GAK (cyclin G-associated kinase). These kinases localize at the centrosome, regulate mitotic progression in response to DNA damage, and cause chromosome instability when their functions are disrupted. Both Lats (Lats1 and Lats2) and GAK form complexes with Mdm2. In turn, Mdm2 controls the stability of p53, which is a transcriptional regulator of the Lats2, cyclin G1 and Mdm2 genes. Thus, the Lats and GAK complexes have intimate correlation in their function (Fig. 1). In addition, to inhibit spontaneous metastasis and the growth of malignant tumors by inhibiting connexin 26, our laboratory has developed safe oleamide-derivative drugs that are associated with few side effects.

Our research subjects are detailed below:
(1) Lats Group:
Lats1 and Lats2, which belong to the Lats kinase family, are highly conserved across species and localize at the centrosome during the cell cycle (Fig. 2). Two miRNAs, miRNA-372 and -373, function as potential novel oncogenes in testicular germ cell tumors by inhibiting LATS2 expression, which suggests that Lats2 is an important tumor suppressor (Voorhoeve et al., Cell, 2006). Lats2 binds Mdm2, thereby inhibiting its E3 ligase activity and activating p53; in turn, p53 rapidly and selectively upregulates Lats2 expression in G2/M cells. This positive feedback loop constitutes a novel checkpoint pathway that plays a critical role in the maintenance of proper chromosome numbers (Aylon et al., Gene Dev., 2006). We have discovered the following:

1. Lats2 knockout mice are embryonic lethal, which indicates the essential role of Lats2 in the development and differentiation of mammalian germ cells.
2. Lats2^-/- mouse embryonic fibroblasts (MEF) display an enhanced growth rate, centrosome fragmentation (Fig. 3), misalignment of the chromosome at M phase, abnormal chromosome segregation, and aberrant cytokinesis. These results indicate the essential role Lats2 plays in proper M phase progression.
3. Aurora-A phosphorylates Lats2 on three distinct serines during mitosis; Lats2 localizes at the centrosome, the mitotic spindle, or the spindle midzone during the cell cycle depending on which site is phosphorylated.
4. Down-regulation of Lats2 by siRNA causes the mislocalization of the chromosomal passenger complex (CPC) during the metaphase/anaphase transition, with the consequence that cytokinesis fails. These observations suggest that the Aurora-A-Lats2-CPC axis is a novel pathway that regulates proper cytokinesis.
5. Lats1/Lats2 knockout mice show arrested development at a very early stage of embryogenesis.

Fig. 2: The structures of the Lats1 and Lats2 proteins are similar.

Fig. 3: Loss of Lats2 leads to centrosome fragmentation.

(2) GAK Group:
GAK is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Unlike neuron-specific auxilin, which plays a similar role in neural cells, GAK has a kinase domain (Fig. 4) whose function has remained unclear. We have discovered the following:

1. GAK forms the KBG（KBG1 and KBG2）complex with PP2A B' γ and cyclin G (cyclin G1 and cyclin G2), which regulate the dephosphorylation activity of PP2A.
2. GAK localizes not only in the cytoplasm but also at the nucleus, where it has two additional functions, namely the maintenance of proper centrosome maturation and mitotic chromosome congression.
3. GAK knockdown by siRNA causes cell cycle arrest at the metaphase, which indicates that GAK is required for proper mitotic progression. This impaired mitotic progression was found to be due to the activation of the spindle assembly checkpoint (SAC), which senses protruding, misaligned, or abnormally condensed chromosomes in knockdown cells. (Fig. 5)
4. CHC is involved in this regulatory process since GAK functions cooperatively with clathrin during mitotic progression as well as during endocytosis. (Fig. 6)

Fig. 4: GAK is similar to auxilin except for bearing a kinase domain.

Fig. 5: GAK knockdown by siRNA (Ki9) generates abnormal chromosomes.

Fig. 6: GAK knockdown by siRNA (Ki9) causes the abnormal localization of CHC.