Satomi Mukai

The tumor suppressor Lats2 is pivotal in Aurora A and Aurora B signaling during mitosis.

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.

N-terminal truncation of Lats1 causes abnormal cell growth control and chromosomal instability.

Summary The tumor suppressors Lats1 and Lats2 are mediators of the Hippo pathway that regulates tissue growth and proliferation. Their N-terminal non-kinase regions are distinct except for Lats conserved domains 1 and 2 (LCD1 and LCD2), which may be important for Lats1/2-specific functions. Lats1 knockout mice were generated by disrupting the N-terminal region containing LCD1 (Lats1&Dgr;N/&Dgr;N). Some Lats1&Dgr;N/&Dgr;N mice were born safely and grew normally. However, mouse embryonic fibroblasts (MEFs) from Lats1&Dgr;N/&Dgr;N mice displayed mitotic defects, centrosomal overduplication, chromosomal misalignment, multipolar spindle formation, chromosomal bridging and cytokinesis failure. They also showed anchorage-independent growth and continued cell cycles and cell growth, bypassing cell-cell contact inhibition similar to tumor cells. Lats1&Dgr;N/&Dgr;N MEFs produced tumors in nude mice after subcutaneous injection, although the tumor growth rate was much slower than that of ordinary cancer cells. Yap, a key transcriptional coactivator of the Hippo pathway, was overexpressed and stably retained in Lats1&Dgr;N/&Dgr;N MEFs in a cell density independent manner, and Lats2 mRNA expression was downregulated. In conclusion, N-terminally truncated Lats1 induced Lats2 downregulation and Yap protein accumulation, leading to chromosomal instability and tumorigenesis.

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis

The tumor suppressor kinases LATS1 and LATS2 (LATS1/2) regulate not only organ size through the Hippo signaling pathway, but also cell-cycle checkpoints and apoptosis via other signaling cascades. We previously reported that LATS1/2 localize to the mitotic apparatus, where they are involved in the phosphorylation and activation of the mitotic kinase Aurora-B; however, the detailed mechanism of LATS1/2 action remains obscure. The activity of Aurora-B is stringently regulated by formation of the chromosomal passenger complex containing the inner centromere protein (INCENP), which leads to appropriate activation of Aurora-B during mitosis and cytokinesis. In this study, we found that LATS1/2 phosphorylated INCENP at S894 in the Thr-Ser-Ser motif. Moreover, the LATS-mediated phosphorylation of S894 was necessary and sufficient for the activation of Aurora-B, which is required for completion of cytokinesis in cells engaged in multipolar division. We propose a novel mechanism for regulation of Aurora-B via INCENP phosphorylation by LATS1/2 during cytokinesis.

Lats1 suppresses centrosome overduplication by modulating the stability of Cdc25B.

Numerical aberration of the centrosome results in chromosome missegregation, eventually leading to chromosomal instability, a hallmark of human tumor malignancy. Large tumor suppressors 1 and 2 (Lats1 and Lats2) are central kinases in the Hippo pathway and regulate development and tumorigenesis by coordinating the balance between cell proliferation and apoptosis. Importantly, Lats1 and Lats2 also play pivotal roles in cell cycle checkpoint and mitosis. The Lats proteins localize at centrosomes, but their centrosomal functions remain elusive. Here, we generated Lats1-null knockout (Lats1−/−) mice and established Lats1-null mouse embryonic fibroblasts (MEFs). In Lats1−/− MEFs, centrosomes were markedly overduplicated, leading to severe mitotic defects such as chromosome missegregation and cytokinesis failure. We also found that Lats1 physically interacts with Cdc25B phosphatase that localizes both at the centrosome and in the nucleus and regulates the linkage between the centrosome cycle and mitotic progression. Although Lats1 did not phosphorylate Cdc25B, loss of Lats1 in MEFs caused abnormal accumulation of Cdc25B protein and hyperactivation of Cdk2 toward nucleophosmin (NPM/B23), one of the licensing factors involved in centriole duplication. Taken together, these data suggest that Lats1 regulates Cdc25B protein level and subsequent Cdk2 activity, thereby suppressing centrosome overduplication during interphase.

Phosphorylation of CHO1 by Lats1/2 regulates the centrosomal activation of LIMK1 during cytokinesis.

Large tumor suppressor 1 and 2 (Lats1/2) regulate centrosomal integrity, chromosome segregation and cytokinesis. As components of the centralspindlin complex, the kinesin-like protein CHO1 and its splicing variant MKLP1 colocalize with chromosome passenger proteins and GTPases and regulate the formation of the contractile ring and cytokinesis; however, the regulatory mechanisms of CHO1/MKLP1 remain elusive. Here, we show that Lats1/2 phosphorylate Ser716 in the F-actin-interacting region of CHO1, which is absent in MKLP1. Phosphorylated CHO1 localized to the centrosomes and midbody, and the actin polymerization factor LIM-kinase 1 (LIMK1) was identified as its binding partner. Overexpression of constitutively phosphorylated and non-phosphorylated CHO1 altered the mitotic localization and activation of LIMK1 at the centrosomes in HeLa cells, leading to the inhibition of cytokinesis through excessive phosphorylation of Cofilin and mislocalization of Ect2. These results suggest that Lats1/2 stringently control cytokinesis by regulating CHO1 phosphorylation and the mitotic activation of LIMK1 on centrosomes.

GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase.

ABSTRACT Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association in vivo. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.

LATS2 Positively Regulates Polycomb Repressive Complex 2

LATS2, a pivotal Ser/Thr kinase of the Hippo pathway, plays important roles in many biological processes. LATS2 also function in Hippo-independent pathway, including mitosis, DNA damage response and epithelial to mesenchymal transition. However, the physiological relevance and molecular basis of these LATS2 functions remain obscure. To understand novel functions of LATS2, we constructed a LATS2 knockout HeLa-S3 cell line using TAL-effector nuclease (TALEN). Integrated omics profiling of this cell line revealed that LATS2 knockout caused genome-wide downregulation of Polycomb repressive complex 2 (PRC2) and H3K27me3. Cell-cycle analysis revealed that downregulation of PRC2 was not due to cell cycle aberrations caused by LATS2 knockout. Not LATS1, a homolog of LATS2, but LATS2 bound PRC2 on chromatin and phosphorylated it. LATS2 positively regulates histone methyltransferase activity of PRC2 and their expression at both the mRNA and protein levels. Our findings reveal a novel signal upstream of PRC2, and provide insight into the crucial role of LATS2 in coordinating the epigenome through regulation of PRC2.