Protein phosphatase type 2A, PP2A, is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp 130 at Ser-782 and degradation of gp 130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp 130. Taken together, present results strongly suggest that degradation of gp 130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp 130 is a potential therapeutic target in cancers.     

Dynamic association of topoisomerase II to the mitotic chromosomes in live cells of Aspergillus nidulans

DNA topoisomerase II (Topo II) is an essential enzyme that catalyzes topological changes of DNA and consists of a major member of mitotic chromosomes. To investigate the dynamic localization of Topo II in nuclei, we engineered the strain of Aspergillus nidulans expressing Topo II fused with green fluorescent protein (GFP). Time-lapse microscopy revealed that the distribution of Topo II-GFP in nuclei varied depending on the cell cycle. In interphase, Topo II-GFP distributed evenly in the nucleoplasm and at the onset of G2 phase became concentrated into nucleolus. During mitosis, Topo II-GFP accumulated on chromosomes, when the chromosomes condensed. In the early mitosis, the Topo II also showed a single or two brighter spots among the fluorescence of clumped chromosomes. The spots once divided into several spots and then concentrated again into a spot per nucleus in the dividing nuclei of anaphase. Along with the subsequent decondensation of chromosomes, Topo II diffused back into nucleoplasm.     

Centromere-specific repetitive sequences from Torenia, a model plant for interspecific fertilization, and whole-mount FISH of its interspecific hybrid embryos

Torenia fournieri is a good model plant to study fertilization in plants because it is easy to observe the fertilization process due to the protruding nature of the embryo sac. To study the location and movement of chromosomes and their centromeres in early embryogenesis of interspecific hybrid plants, we isolated two families of centromere-specific tandem repetitive sequences from T. fournieri and T. bailonii, and named them the "TCEN-family" and "BCEN-family", respectively. Both sequences consisted of a repeat unit of 52 bp located in the pericentric and centric heterochromatins. All signals of both sequences were prominent, but their intensity varied among the chromosomes. DNA-blot hybridization indicated the presence of similar sequences of TCEN-family in T. concolor, N. caerulea, and "Summer Wave", whereas the BCEN-family was found only in T. bailonii, thus indicating the wide or specific distribution of their repetitive families observed. We also applied whole-mount FISH to the interspecific hybrid embryos by using TCEN- and BCEN-family sequences as probes. Our results suggest that whole-mount FISH with the species-specific centromere sequences as probes is an ideal method to analyze the dynamics of chromosomes and centromeres in interspecific fertilization and early embryogenesis.     

Serum evaluation of the balance between soluble interleukin-2 and interleukin-4 receptors

To elucidate the usefulness of the simultaneous analysis of the multiple kinds of soluble cytokine receptors, we determined both the soluble interleukin 2 receptor (sIL-2R, Th1-type cytokine receptor) and the soluble interleukin 4 receptor (sIL-4R, Th2-type cytokine receptor) levels in the sera of healthy subjects as reference values and preliminarily applied to evaluate the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameter of the severity. Both sIL-2R and sIL-4R levels in the sera of healthy children were significantly higher than those of healthy adults (p<0.01). The serum sIL-2R level of the patients with severe HUS (n=4) was higher than that of the patients with mild/moderate HUS (n=6) at the initial stage (p<0.01) or healthy children (n=51, p<0.01). Whereas, the serum sIL-4R level of both the severe and mild/moderate groups was lower than that of the healthy control children, although there was no significant difference among the three groups. Namely, the soluble receptor balance (sIL-2R/sIL-4R) in the patients with severe HUS may shift. We considered that the evaluation of the balance between soluble cytokine receptors might be informative for the evaluation of the immune states, as well as the conventional cytokine balance (Th1/Th2).     

Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.     

Prediction of folding pathway and kinetics among plant hemoglobins using an average distance map method

Computational methods, such as the ADM (average distance map) method, have been developed to predict folding of homologous proteins. In this work we used the ADM method to predict the folding pathway and kinetics among selected plant nonsymbiotic (nsHb), symbiotic (Lb), and truncated (tHb) hemoglobins (Hbs). Results predicted that (1) folding of plant Hbs occurs throughout the formation of compact folding modules mostly formed by helices A, B, and C, and E, F, G, and H (folding modules A/C and E/H, respectively), and (2) primitive (moss) nsHbs fold in the C-->N direction, evolved (monocot and dicot) nsHbs fold either in the C-->N or N-->C direction, and Lbs and plant tHbs fold in the C-->N direction. We also predicted relative folding rates of plant Hbs from qualitative analyses of the stability of subdomains and classified plant Hbs into fast and moderate folding. ADM analysis of nsHbs predicted that prehelix A plays a role during folding of the N-terminal domain of Ceratodon nsHb, and that CD-loop plays a role in folding of primitive (Physcomitrella and Ceratodon) but not evolved nsHbs. Modeling of the rice Hb1 A/C and E/H modules showed that module E/H overlaps to the Mycobacterium tuberculosis HbO two-on-two folding. This observation suggests that module E/H is an ancient tertiary structure in plant Hbs.     

Ubiquitin-interacting motifs of Epsin are involved in the regulation of insulin-dependent endocytosis

Epsin is a key molecule in receptor-mediated endocytosis. Epsin is phosphorylated and ubiquitinated, and these post-translational modifications are necessary for the regulation of endocytosis. Since human Epsin (hEpsin) has two ubiquitin-interacting motifs (UIMs), we investigated the roles of these UIMs in endocytosis. hEpsin formed a complex with ubiquitinated proteins but did not bind to monoubiquitin. Neither of the two UIMs of hEpsin alone was sufficient to form a complex with ubiquitinated proteins: both UIMs were necessary. Mutations of Asp209 and Asp210 to Ala in UIM (hEpsinDA) abolished the binding activity of hEpsin to ubiquitinated proteins. However, hEpsinDA interacted with Eps15, POB1, and AP-2, which are involved in receptor-mediated endocytosis, as efficiently as wild-type hEpsin. Expression of hEpsinDA in CHO-IR cells affected neither the binding of insulin to nor insulin-dependent autophosphorylation of its receptor. Expression of wild-type hEpsin inhibited the internalization of insulin, whereas that of hEpsinDA did not. These results suggest that the UIM motifs of hEpsin interact with proteins modified with ubiquitin, and that the complex formation is involved in insulin-dependent receptor endocytosis.     

Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.