Shigeo Ohno

We previously reported that aberrant expression of atypical protein kinase C λ/ι (aPKCλ/ι) in low-grade squamous intraepithelial uterine cervix lesions was associated with an increased risk of progression to higher grade. This study aimed to investigate aPKCλ/ι expression patterns in cervical squamous cell carcinoma (SCC) and its association with disease progression. We immunohistochemically assessed aPKCλ/ι expression in 168 SCC samples and 13 normal uterine cervix samples. In 69.0% of SCC cases, aPKCλ/ι was expressed more abundantly than in normal epithelium, but there was no significant association between aPKCλ/ι intensity and disease progression (P=0.087, Cochran-Mantel-Haenszel test). aPKCλ/ι in normal cervical epithelium was confined to the cytoplasm or intercellular junctions. In contrast, aPKCλ/ι was predominantly localized within the nucleus in 36.9% of SCC samples (P<0.001, χ2 test), and the prevalence was significantly increased relative to advanced tumor stage (P<...

Atypical protein kinase C λ/ι (aPKCλ/ι) is a regulator of epithelial cellular polarity. It is also overexpressed in several cancers and functions in cell proliferation and invasion. Therefore, we hypothesized that aPKCλ/ι may be involved in development and progression of cervical intraepithelial neoplasia (CIN), the precancerous disease of cervical cancer induced by human papillomavirus. To do this, we investigated the relationship between aPKCλ/ι expression and CIN. aPKCλ/ι expression level and subcellular localization were assessed in 192 CIN biopsy samples and 13 normal epithelial samples using immunohistochemistry. aPKCλ/ι overexpression (normal epithelium, 7.7%; CIN1, 41.7%; CIN2/3, 76.4%) and aPKCλ/ι nuclear localization (normal epithelium, 0.0%; CIN1, 36.9%; CIN2/3, 78.7%) were higher in CIN samples than normal samples (P<0.05), suggesting that CIN grade is related to aPKCλ/ι overexpression and nuclear localization. Then, 140 CIN cases were retrospectively analyzed for 4-y...

p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKCλ, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKCλ associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKCλ results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKCλ, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKCλ, however, fails to activate p70 S6K. These results suggest that PKCλ is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression.

Presenilin 1 (PS1) is the causative gene for an autosomal dominant familial Alzheimer's disease (AD) mapped to chromosome 14. Here we show that QM/Jun-interacting factor (Jif)-1, a negative regulator of c-Jun, is a candidate to mediate the function of PS1 in the cell. We screened for proteins that bind to PS1 from a human embryonic brain cDNA library using the two-hybrid method and isolated one clone encoding the QM/Jif-1 gene. The binding of QM/Jif-1 to full-length PS1 was confirmed in vitro by pull-down assay, and in vivo by immunoprecipitation assays with human samples, including AD brains. Immunoelectronmicroscopic analysis showed that QM/Jif-1 and PS1 are colocalized at the endoplasmic reticulum, and the nuclear matrix in human brain neurons. Chloramphenicol acetyltransferase assays in F9 cells showed that PS1 suppresses transactivation by c-Jun/c-Jun but not by c-Jun/c-Fos heterodimers, consistent with the reported function of QM/Jif-1. By monitoring fluorescent recombinan...

Background: Epithelial cell polarity is guided by highly conserved polarity proteins. Especially aPKC lambda/iota is a pivotal regulator of cell polarity, and related to oncogenecity. LST is a flat-type colorectal tumor that is macroscopic morphologic concept in contrast to a protruded-type tumor. Cancer in LST also show a superficial-replacing growth and less invasive in an early stage of cancer. Previously, we showed expression of aPKC l/i is important for determination of structures in LSTs. In the present study, we examined morphologic characteristics of adenomatous and cancerous lesions of LSTs, compared with those of protruded-type tumors or flat-depressed type tumors from expression of 4 factors. Material & Methods: In these studies, 37 flat type colorectal tumors and 20 polypoid-type colorectal tumors resected endoscopically or surgically in the Yokohama City University Hospital from 1998 to 2007 were examined. Flat type tumors included 15 LST adenomas, 9 LSTs with cancer in...

Background: LSTs grow superficially along the colonic wall but are short in height despite diameters larger than 10 mm. In our investigation, LSTs maintain cell polarity in spite of neoplasms comparing with polypoid-type tumor. In the present study, we examined the microscopic morphologies and functional characteristics of LSTs using immunohistochemical studies. Material and Methods: 30 LSTs and 20 polypoid-type colorectal tumors resected endoscopically or surgically in the department of Gastroenterological Surgery of Yokohama City University Hospital from 1998 to 2007, were examined. Localization of beta-catenin or E-cadherin, conservation of basement membrane and aPKCl/i were investigated by immunohistochemistry. Results: The expression of beta-catenin was of the preserved type in 7 (87.5%) of the LST adenomas, 11 (100%) of the polypoid-type adenomas and 7 (77.8%) of the LST intramucosal cancers. The expression of beta-catenin was of the nucleic type in 5 (55.6%) and the preserved type in only 2 (22.2%) of the polypoid-type cancers in adenoma. The expression of E-cadherin was also of the cytoplasmic or preserved type in all cases of the LST adenomas and polypoid-type adenomas and 8 (88.9%) of the LST intramucosal cancers. The expression of E-cadherin was of the lost type in none (0%) of the LST adenomas and polypoid-type adenomas while it was of the lost type in 5 (55.6%) of the polypoid-type cancers in adenoma. The expression of type IV collagen was of the continuous type in 8 (100%) of the LST adenomas and 6 (66.7%) of the LST intramucosal cancers. The expression of type IV collagen was discontinuous in 8 (72.7%) of [the] polypoid-type adenomas and lost in 7 (77.8%) of the polypoid-type cancers in adenoma. The expression of type IV collagen was of the lost type in all cases of LST invasive cancers with adenoma. Conclusion: In aPKC lambda/iota e xpression, >90% of adenoma was 1+ or 2+. 87% of LST adenomas were 1+ and 45% of polypoid-type adenomas were 2+. About 70% of cancer in polypoid-type adenoma and invasive cancer of LSTs were 3+. On the other hands, about 60% of LST intramucosal cancers were 1+. LST adenomas or LST cancer in situ showed weaker expression of aPKC than polypoid-type adenomas or cancers. In the site of strong expression of aPKCl ambda/iotab eta-catenin was expressed in nucleus and in the site of weak expression of aPKC lambda/iota, beta-catenin was expressed in cytoplasm and cell membrane Conclusions: In the present study, we showed that in LSTs, not only adenomatous lesions, but also intramucosal cancerous lesions, maintained the characteristics of normal epithelial cells in the expression of beta-catenin, E-cadherin and Type IV collagen. They also showed weak expression of aPKC lambda/iota. (Supported by the grants of Japanese Ministry of Education, Culture, Sports and Science) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5176.

Hydrolysis of inositol phospholipids by receptor stimulation activates two separate signaling pathways, one leading to the activation of protein kinase C (C kinase) via formation of diacylglycerol. The other is the inositol trisphosphate (IP3)/Ca2+ pathway and a major downstream kinase which is activated is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). To examine signaling pathways of C kinase and CaM kinase II to the cytoskeletal protein vimentin, we prepared monoclonal antibodies YT33 and MO82 which recognize the phosphorylation state of vimentin by C kinase and by CaM kinase II, respectively. Ectopic expression of constitutively active C kinase or CaM kinase II in primary cultured astrocytes by microinjection of the corresponding expression vectors induced phosphorylation of vimentin at each specific phosphorylation site, followed by reorganization of vimentin filament networks. In contrast, simultaneous activation of C kinase and CaM kinase II by inositol phosphol...

hSMG-1 is a member of the phosphoinositide 3 kinase-like kinase (PIKK) family with established roles in nonsense-mediated decay (NMD) of mRNA containing premature termination codons and in genotoxic stress responses to DNA damage. We report here a novel role for hSMG-1 in cytoplasmic stress granule (SG) formation. Exposure of cells to stress causing agents led to the localization of hSMG-1 to SG, identified by colocalization with TIA-1, G3BP1, and eIF4G. hSMG-1 small interfering RNA and the PIKK inhibitor wortmannin prevented formation of a subset of SG, while specific inhibitors of ATM, DNA-PK(cs), or mTOR had no effect. Exposure of cells to H(2)O(2) and sodium arsenite induced (S/T)Q phosphorylation of proteins. While Upf2 and Upf1, an essential substrate for hSMG-1 in NMD, were present in SG, NMD-specific Upf1 phosphorylation was not detected in SG, indicating hSMG-1's role in SG is separate from classical NMD. Thus, SG formation appears more complex than originally envisage...

ABSTRACTDuring brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis crucially affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to the neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation at the expense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of hedgehog signaling in a smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of smoothened and an inhibitor of smoothened ameliorated the hyperproliferation of NPCs in the ...

s / Neuroscience Research 58S (2007) S1–S244 S239 P3-j3Ø Behavioral and histological examinations in TR-alpha or TR-beta deficient mice Miyuki Sadamatsu1, Hirohiko Kanai2, Nobumasa Kato3 1 Department of Psychiatry, Nara Medical University, Kashihara, Japan; 2 Department of Psychiatry, Shiga University Medical Science, Otsu, Japan; 3 Department of Psychiatry, Faculty of Medicine, Showa University, Tokyo, Japan Thyroid functions are well known to participate in neuronal development after birth in mice. The drug-induced hypothyroidism in a early period of the neuronal development results in permanent behavioral abnormality, including memory disturbance and hyperactivity. However, the relevance of the memory disturbance and the hyperactivity to thyroid hormone receptors, TR-alpha and TR-beta, remains unknown. To address this issue, we performed histological examination and behavioral experiments, including open field and radial maze in TR-alpha and TR-beta deficient mice. In open field experiment, the TR-alpha deficient mice exhibited the tendency of hyperactivity, but unexpectedly we did not observed any gross behavioral abnormalities in TR-beta deficient mice. In this study, we explored the possible involvement of the thyroid hormone signaling in the development of the brain functions after birth. P3-j32 CtBPs are binding proteins of the novel isoform of FoxP2 lacking forkhead domain Eriko Fujita1, Ayumi Matsuzaki1,2, Yuko Tanabe1, Tadashi Kasahara2, Mariko Momoi3, Takashi Momoi1 1 National Institute of Neuroscience, NCNP, Japan; 2 Kyoritsu University of Pharmacy, Tokyo, Japan; 3 Jichi Medical University, Tochigi, Japan The mutations of FOXP2 have been detected in the family of the speech/language disorder. Recently, we identified the forkhead nuclear localization domains that contribute to the cellular distribution of FOXP2. Nuclear localization of FOXP2 depended on two distally separated nuclear localization signals in the forkhead domain. In contrast with wildtype, the mutated FOXP2s are located in the cytoplasm. However, we also identified the novel isofrom lacking forkhead domain located in the cytoplasm of Perkinje cells (P2-P10). To examine the biological role of the novel isoform, we tried to isolate the proteins binding with the novel isoform of FOXP2 by yeast-two-hybrid system. Here we show that this isoform specifically binds with CtBPs with multifuctions such as repressor, golgi fission, and synapticvesicle fusion. The novel isoform may be involved in the synaptic function via binding with CtBPs. Research fund: KAKENHI (18700333). P3-j33 Identification of a novel isoform of Foxp2 located in the cytoplasm of mouse developing Purkinje cells Ayumi Matsuzaki1,2, Yuko Tanabe1, Eriko Fujita1, Tadashi Kasahara2, Takashi Momoi1 1 Department of Inherited Metabolic Diseases, NCNP, Tokyo, Japan; 2 Department of Biochemistry, Kyoritsu University of Pharmacy, Tokyo, Japan FOXP2 belongs to a newly identified subfamily of Forkhead box (FOX)transcription factors. The mutations of FOXP2 have been detected in the family of the speech/language disorder, suggesting that FOXP2 is involved in acquiring human speech/language ability. In contrast with wild type, the mutated FOXP2s are located in the cytoplasm. In the present study, we identified the forkhead nuclear localization domains that contribute to the cellular distribution of FOXP2. Nuclear localization of FOXP2 depends on two distally separated nuclear localization signals in the forkhead domain. Furthermore, we identified the novel isofrom lacking forkhead domain located in the cytoplasm during mouse brain development. In the mouse cerebellum, this novel isoform was mainly detected in the cytoplasm of the Purkinje cells (P2-P10), but not in those of P15-P22. This novel isoform may be involved in the acquiring mouse “singing” ability related to the human speech/language ability. P3-j34 Brain proteins with PDZ domains associated with RA175/SynCAM Yuko Tanabe1, Ayumi Matsuzaki1, Eriko Fujita1, Giulo Piluso2, Shigeo Ohno3, Shouichi Ishiura4, Alaa Hussein5, Vencenzo Nigro2, Takashi Momoi1 1 National Institute of Neuroscience,Tokyo, Japan; 2 Seconda University, Napoli, Italy; 3 Yokohama City University Graduate School of Medical Science; 4 University, Tokyo; 5 University British Columbia, Vancouver, BC, Canada Association of neuroligins with PSD-95 and Shank controls the balance between neuronal excitation and inhibition. Mutations of neuroligin-3 and Shank-3 have been detected in the patient of Autism. RA175/SynCAM, homophilic cell adhesion molecule with PDZ binding domain at Cterminal, is also involved in the formation of functional synapse. RA175/SynCAM interacts with CASK at presynapse, but little is known about the binding molecules at postsynapse. We examined the RA175Cterminal binding protein in mouse brain by yeast two hybrid analysis and pull-down assay. Unlike neuroligin, RA175 directly band with Par-3 and RA175 had a complex with Shank but not with…

Cell polarity is essential for various asymmetric cellular events, where the partitioning defective (PAR) protein, PAR3, plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border such as the tight junction in vertebrates and functions as an apical determinant. Although there is much information about the regulators of PAR3 localization, the mechanism involved in PAR3 concentration and localization to the specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2, a stimulator of PAR3 localization, can link PAR3 and protein phosphatase 1 (PP1). The ASPP2–PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of ...

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion...

cDNA clones coding for novel protein kinase C delta (nPKC delta) were isolated from a mouse brain cDNA library. Mouse nPKC delta consists of 674 amino acid residues and has sequence identity of 95% with rat nPKC delta. Antiserum raised against a C-terminal peptide of rat nPKC delta identified a 79-kDa protein in COS cells transfected with a mouse nPKC delta cDNA expression plasmid. nPKC delta expressed in COS1 cells had phorbol-ester-binding activity and protein kinase activity in a phorbol-ester- or diacylglycerol-dependent manner, like conventional protein kinase C (cPKC) isozymes and nPKC epsilon. However, nPKC delta, like nPKC epsilon, is not activated by Ca2+, a known activator of cPKCs, and requires lower concentrations of Mg2+ for full activation than cPKCs. Moreover, apparent kinetic constants for synthetic oligopeptides (MBP4-14, EGFR peptide and epsilon-peptide) were quite different between nPKC delta and cPKC in two different conditions. Among various phospholipids tested, phosphatidylinositol is the most potent activator of nPKC delta, in clear contrast to cPKCs and nPKC epsilon. Limited proteolysis of nPKC delta generated a C-terminal active fragment with a cofactor-independent kinase activity. Northern blot analysis indicated that nPKC delta, like cPKC alpha, is widely distributed in almost all the tissues and cells examined and, in some cases such as fibroblast cells, exists as a major PKC type. These results suggest that nPKC delta is involved in fundamental cellular functions regulated by diacylglycerols and mimicked by phorbol esters.

In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKCλ, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKCλ knockout mice, neuroepithelial cells of the neocortical region lost aPKCλ protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKCλ is indispensable for the maintenance of adherens junctions and may function i...

The evolutionarily conserved polarity proteins PAR-3, atypical protein kinase C (aPKC) and PAR-6 critically regulate the apical membrane development required for epithelial organ development. However, the molecular mechanisms underlying their roles remain to be clarified. We demonstrate that PAR-3 knockdown in MDCK cells retards apical protein delivery to the plasma membrane, and eventually leads to mislocalized apical domain formation at intercellular regions in both two-dimensional and three-dimensional culture systems. The defects in PAR-3 knockdown cells are efficiently rescued by wild-type PAR-3, but not by a point mutant (S827/829A) that lacks the ability to interact with aPKC, indicating that formation of the PAR-3–aPKC–PAR-6 complex is essential for apical membrane development. This is in sharp contrast with tight junction maturation, which does not necessarily depend on the aPKC–PAR-3 interaction, and indicates that the two fundamental processes essential for epithelial pol...

The mammalian protein ASIP/PAR-3 interacts with atypical protein kinase C isotypes (aPKC) and shows overall sequence similarity to the invertebrate proteins C. elegans PAR-3 and Drosophila Bazooka, which are crucial for the establishment of polarity in various cells. The physical interaction between ASIP/PAR-3 and aPKC is also conserved in C. elegans PAR-3 and PKC-3 and in Drosophila Bazooka and DaPKC. In mammals, ASIP/PAR-3 colocalizes with aPKC and concentrates at the tight junctions of epithelial cells, but the biological meaning of ASIP/PAR-3 in tight junctions remains to be clarified. In the present study, we show that ASIP/PAR-3 staining distributes to the subapical domain of epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy revealed that...

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3β activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3β complexes controls local GSK3β activity and thereby regulates endothelial planar polarity. The spatial information for GSK3β activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced a...