Clathrin heavy chain phosphorylated at T606 plays a role in proper cell division.

Clathrin regulates mitotic progression, in addition to membrane trafficking. However, the detailed regulatory mechanisms of clathrin during mitosis remain elusive. Here, we demonstrate novel regulation of clathrin during mitotic phase of the cell cycle. Clathrin heavy chain (CHC) was phosphorylated at T606 by its association partner cyclin G-associated kinase (GAK). This phosphorylation was required for proper cell proliferation and tumor growth of cells implanted into nude mice. Immunofluorescence analysis showed that the localization of CHC-pT606 signals changed during mitosis. CHC-pT606 signals localized in the nucleus and at the centrosome during interphase, whereas CHC signals were mostly cytoplasmic. Co-immunoprecipitation suggested that CHC formed a complex with GAK and polo-like kinase 1 (PLK1). Depletion of GAK using siRNA induced metaphase arrest and aberrant localization of CHC-pT606, which abolished Kiz-pT379 (as a phosphorylation target of PLK1) signals on chromatin at metaphase. Taken together, we propose that the GAK_CHC-pT606_PLK1_Kiz-pT379 axis plays a role in proliferation of cancer cells.

LATS1/2 kinases trigger self-renewal of cancer stem cells in aggressive oral cancer.

Cancer stem cells (CSCs), which play important roles in tumor initiation and progression, are resistant to many types of therapies. However, the regulatory mechanisms underlying CSC-specific properties, including self-renewal, are poorly understood. Here, we found that LATS1/2, the core Hippo pathway-kinases, were highly expressed in the oral squamous cell carcinoma line SAS, which exhibits high capacity of CSCs, and that depletion of these kinases prevented SAS cells from forming spheres under serum-free conditions. Detailed examination of the expression and activation of LATS kinases and related proteins over a time course of sphere formation revealed that LATS1/2 were more highly expressed and markedly activated before initiation of self-renewal. Moreover, TAZ, SNAIL, CHK1/2, and Aurora-A were expressed in hierarchical, oscillating patterns during sphere formation, suggesting that the process consists of four sequential steps. Our results indicate that LATS1/2 trigger self-renewal of CSCs by regulating the Hippo pathway, the EMT, and cell division.

Late cornified envelope 1C (LCE1C), a transcriptional target of TAp63 phosphorylated at T46/T281, interacts with PRMT5.

p63, a transcriptional factor that belongs to the p53 family, regulates epidermal differentiation, stemness, cell death, tumorigenesis, metastasis, and senescence. However, its molecular mechanism remains elusive. We report here that TAp63 phosphorylated at T46/T281 specifically upregulates the late cornified envelope 1C (LCE1C) gene that is essential at a relatively late stage of epithelial development. We identified these phosphorylation sites during a search for the targets of Cyclin G-associated kinase (GAK) in vitro. LCE1C was drastically upregulated by doxycycline-dependent expression of Myc-TAp63 wild-type protein. Luciferase reporter assays using the promoter region of the LCE1C gene confirmed that the phosphorylations of TAp63-T46/T281 contributed to full transcriptional activation of the LCE1C gene. LCE1C interacted with protein arginine methyltransferase 5 (PRMT5) and translocated it from the nucleus to the cytoplasm. Mass spectrometry and co-immunoprecipitation identified importin-α as one of the association partners of LCE1C. In summary, we propose that the GAK_TAp63-pT46/pT281_LCE1C axis plays an important role in preventing the nuclear function of PRMT5.

ELAS1 induces apoptotic death in adenocarcinoma DU145 and squamous-cell carcinoma SAS cancer cells, but not in normal KD cells.

We previously reported that an ELAS1 peptide containing 29 amino acids induces apoptotic death in U2OS human osteosarcoma cells following DNA double-strand break insults. Here, we show that ELAS1 also caused apoptosis in prostate adenocarcinoma DU145 cells and tongue squamous-cell carcinoma SAS cells. ELAS1 appears to be safe because it induced apoptosis only in cancer cells, not in normal KD cells. Because the effect of ELAS1 is dependent on increased stability of p53 and enhanced phosphorylation of p53-S46, we exogenously expressed wild-type p53 protein to fully promote ELAS1-mediated induction of apoptosis in SAS cells. Interestingly, simultaneous expression of Myc-ELAS1 and FLAG-p53 mediated by an internal ribosome entry site efficiently induced apoptosis in SAS cells. Moreover, we prepared a recombinant adenovirus that simultaneously expressed Myc-ELAS1 and FLAG-p53. This adenovirus also killed SAS cells, as determined by a cell viability assay, in the presence of camptothecin, an inducer of DNA double-strand breaks. Moreover, nude mice harboring Myc-ELAS1-expressing SAS cells lived longer than mice harboring Myc-vector-expressing SAS cells, suggesting the usefulness of ELAS1 in vivo. Notably, Cy5-tagged ELAS1-t, which contained only ten amino acids, also efficiently induced apoptosis in both DU145 and SAS cells, suggesting the usefulness of ELAS1-t as a peptide. Taken together, our results suggest that ELAS1 is therapeutically useful as a peptide drug.

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

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.

Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B'γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan-Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Microarray and whole-exome sequencing analysis of familial Behçet's disease patients.

Behçet's disease (BD), a chronic systemic inflammatory disorder, is characterized by recurrent oral and genital mucous ulcers, uveitis, and skin lesions. We performed DNA microarray analysis of peripheral blood mononuclear cell (PBMC) mRNA from 41 Japanese BD patients and revealed elevated levels of interleukin (IL) 23 receptor (IL23R) mRNA in many BD patients. DNA sequencing around a SNV (Rs12119179) tightly linked to BD revealed an elevated frequency of the C genotype, consistent with a previous report that IL23R is a susceptibility locus for BD. Notably, four of these BD patients are members of familial BD; a whole-exome sequencing (WES) of these BD patients identified 19 novel single-nucleotide variations (SNVs) specific to these patients. They include heterozygous SNVs in the genes encoding IL-1 receptor-associated kinase 4 (IRAK4), nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 14 (NRP14) and melanoma antigen-encoding gene E2 (MAGEE2); IRAK4 harbors a missense mutation, whereas NRP14 and MAGEE2 harbor nonsense mutations. These SNVs may serve as genetic markers that characterize BD.

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.