Yi-Ren Hong

Differential expression of Wnt genes, β-catenin and E-cadherin in human brain tumors

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. E-cadherin was discovered as a protein associated with beta-catenin which plays a crucial role in cell-cell adhesion. To further understand the molecular basis of Wnt signaling pathway and E-cadherin in brain tumorigenesis, the expression of four Wnt genes (Wnt1, Wnt5a, Wnt10b and Wnt13) and E-cadherin were analyzed by reverse transcriptase-polymerase chain reaction. In addition, their downstream effector, beta-catenin, was also investigated. The results showed that the expression of Wnt5a (41/45), Wnt10b (37/45), and Wnt13 (35/45) were found in brain tumors, whereas Wnt1 (6/45) was shown to be less related. Interestingly, E-cadherin was only expressed in a few cases of astrocytoma (2/16), whereas it was expressed in most meningioma (14/15) and pituitary adenoma tumors (12/14). There was no apparent difference of beta-catenin expression profile in brain tumors; however, the sequencing data of beta-catenin showed two mutations on speculative phosphorylation sites, S73F and S23G in astrocytoma. Furthermore, an in vitro functional assay showed that S73F and S23G mutants of beta-catenin did not affect transcriptional activity in TCF-4-leuciferase reporter construct, suggesting that they may need more complex factors to participate in astrocytoma. Taken together, our data suggest that the mutations of beta-catenin together with E-cadherin and Wnt signaling might be involved in brain tumorigenesis.

PhosphoPOINT: a comprehensive human kinase interactome and phospho-protein database

MOTIVATION To fully understand how a protein kinase regulates biological processes, it is imperative to first identify its substrate(s) and interacting protein(s). However, of the 518 known human serine/threonine/tyrosine kinases, 35% of these have known substrates, while 14% of the kinases have identified substrate recognition motifs. In contrast, 85% of the kinases have protein-protein interaction (PPI) datasets, raising the possibility that we might reveal potential kinase-substrate pairs from these PPIs. RESULTS PhosphoPOINT, a comprehensive human kinase interactome and phospho-protein database, is a collection of 4195 phospho-proteins with a total of 15 738 phosphorylation sites. PhosphoPOINT annotates the interactions among kinases, with their down-stream substrates and with interacting (phospho)-proteins to modulate the kinase-substrate pairs. PhosphoPOINT implements various gene expression profiles and Gene Ontology cellular component information to evaluate each kinase and their interacting (phospho)-proteins/substrates. Integration of cSNPs that cause amino acids change with the proteins with the phosphoprotein dataset reveals that 64 phosphorylation sites result in a disease phenotypes when changed; the linked phenotypes include schizophrenia and hypertension. PhosphoPOINT also provides a search function for all phospho-peptides using about 300 known kinase/phosphatase substrate/binding motifs. Altogether, PhosphoPOINT provides robust annotation for kinases, their downstream substrates and their interaction (phospho)-proteins and this should accelerate the functional characterization of kinomemediated signaling. AVAILABILITY PhosphoPOINT can be freely accessed in http://kinase. bioinformatics.tw/. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3β

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle☆

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.

A novel ninein‐interaction protein, CGI‐99, blocks ninein phosphorylation by GSK3β and is highly expressed in brain tumors

To explore more hNinein interacting proteins, the yeast two‐hybrid screening using ninein C‐terminal domain as bait protein was performed. One novel gene, CGI‐99, was demonstrated to associate with hNinein in the yeast two‐hybrid method and in vitro GST pull‐down assay. Molecular characterization also showed that CGI‐99 possessed a transcriptional activity at the N‐terminal. In addition, CGI‐99 formed a dimer with the C‐terminal, which overlapped with hNinein binding site. In kinase assay, CGI‐99 binds to hNinein and completely blocks the phosphorylation of hNinein by GSK3β. Moreover, CGI‐99 was highly expressed in all brain tumors which is in agreement with the Northern blot analysis. Taken together, we have isolated a novel protein CGI‐99, which may be involved in the functional regulation of human ninein in the centrosome structure and may also be important in brain development and tumorigenesis.

Glycogen synthase kinase 3beta interacts with and phosphorylates the spindle-associated protein astrin.

Emerging evidence shows that glycogen synthase kinase 3β (GSK3β) is involved in mitotic division and that inhibiting of GSK3β kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3β involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3β interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3β and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3β impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3β. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3β. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3β-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3β differentially increases GSK3β-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3β interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3β-Astrin complex may also facilitate further physiological and pathological phosphorylation.

Differential expression of centrosomal proteins at different stages of human glioma

BackgroundHigh-grade gliomas have poor prognosis, requiring aggressive treatment. The aim of this study is to explore mitotic and centrosomal dysregulation in gliomas, which may provide novel targets for treatment.MethodsA case-control study was performed using 34 resected gliomas, which were separated into low- and high-grade groups. Normal human brain tissue was used as a control. Using immunohistochemical analysis, immunofluorescent microscopy, and RT-PCR, detection of centrins 1 and 2, γ-tubulin, hNinein, Aurora A, and Aurora B, expression was performed. Analysis of the GBM8401 glioma cell line was also undertaken to complement the in vivo studies.ResultsIn high-grade gliomas, the cells had greater than two very brightly staining centrioles within large, atypical nuclei, and moderate-to-strong Aurora A staining. Comparing with normal human brain tissue, most of the mRNAs expression in gliomas for centrosomal structural proteins, including centrin 3, γ-tubulin, and hNinein isoforms 1, 2, 5 and 6, Aurora A and Aurora B were elevated. The significant different expression was observed between high- and low-grade glioma in both γ-tubulin and Aurora A mRNA s. In the high-grade glioma group, 78.6% of the samples had higher than normal expression of γ-tubulin mRNA, which was significantly higher than in the low-grade glioma group (18.2%, p < 0.05).ConclusionsMarkers for mitotic dysregulation, such as supernumerary centrosomes and altered expression of centrosome-related mRNA and proteins were more frequently detected in higher grade gliomas. Therefore, these results are clinically useful for glioma staging as well as the development of novel treatments strategies.

Rac2 expression and mutation in human brain tumors.

SummaryBackground. Rac1 and Rac2 are interchangeable in NADPH oxidase activation. Rac1 plays an important role in regulating nuclear signalling and in the activation of transcriptional factors that regulate gene expression and cell growth. Our previous study observed mutation in effector region of Rac1 gene in brain tumors. Little is known about the expression and mutation of Rac2 in human brain tumors.Method. We examined the expression of Rac2 by using reverse transcriptase-polymerase chain reaction (RT-PCR) and northern blot analysis and the mutation of Rac2 gene by using DNA sequence analysis.Findings. The decreased expression of Rac2 was found in 15 cases (57.7%) including 8 of 10 astrocytomas, 2 of 8 meningiomas, and 5 of 8 pituitary adenomas. Two of 13 cases with decreased expression of Rac2 had gene mutation. Only two of 26 cases had Rac2 overexpression in which no Rac2 gene mutation was found. Four of 8 cases with normal Rac2 expression had Rac2 gene mutation. The site of Rac2 gene mutation had no hot spots and was not concentrated in the effector region.Conclusions. Our results showed a low frequency of mutation and no hot spots of mutation in Rac2 gene in brain tumors, suggesting a decreased possibility of Rac2 in the brain tumorigenesis. The role of high frequency of decreased Rac2 expression in brain tumors, particularly in malignant astrocytomas, needs further investigations to be elucidated.

17β-Estradiol Activates Adenosine A2a Receptor After Subarachnoid Hemorrhage

BACKGROUND Our previous study showed that 17beta-estradiol (E2) and an adenosine A(2A) receptor (AR-A(2A)) agonist could attenuate subarachnoid hemorrhage (SAH)-induced cerebral vasospasm via preventing the augmentation of iNOS expression and preserving the normal eNOS expression. This study tests the hypothesis that E2 attenuates SAH-induced vasospasm and apoptosis by activating adenosine AR-A(2A) and extracellular signal-regulated kinase 1 and 2 (ERK1/2), and by altering antiapoptotic and proapoptotic protein expression (Bcl-2 and Bax, respectively). MATERIALS AND METHODS The two-hemorrhage SAH model in rat was used. Animals were treated with E2 with or without a nonselective estrogen receptor (ER) antagonist (ICI182,780). The cross sectional areas of the basilar artery and terminal dUTP nick-end labeling (TUNEL) were used to determine the degree of vasospasm and apoptosis, respectively. The expressions of Bcl-2, Bax, AR-A(2A), and ERK1/2 in the cerebral cortex, hippocampus, and dentate gyrus were investigated. RESULTS E2 significantly attenuated vasospasm. Seven days after the first SAH, TUNEL scores were significantly increased, and protein levels of AR-A(2A), ERK1/2, and Bcl-2 were significantly decreased in the dentate gyrus only but not in the cortex and hippocampus. These changes were reversed by E2 while ICI182,780 abrogated the antiapoptotic and anti-spastic effects of E2. The expression of Bax did not change in the dentate gyrus after SAH with or without treatment. CONCLUSIONS The down-regulated AR-A(2A) and ERK may play a role in vasospasm and apoptosis after SAH. The beneficial effect of E2 in the attenuating SAH-induced vasospasm and apoptosis may be due to an increased expression of AR-A(2A) and ERK via ER-dependent mechanisms. These data may support further investigation of E2 in the treatment of SAH in humans.

GSK3β regulates Bcl2L12 and Bcl2L12A anti-apoptosis signaling in glioblastoma and is inhibited by LiCl

BCL2L12 has been reported to be involved in post-mitochondrial apoptotic events in glioblastoma, but the role of BCL2L12A, a splicing variant of BCL2L12, remains unknown. In this study, we showed that BCL2L12 and BCL2L12A were overexpressed in glioblastoma multiforme (GBM). Large-scale yeast two-hybrid screening showed that BCL2L12 was a GSK3b binding partner in a testis cDNA library. Our data demonstrated that GSK3b interacts with BCL2L12 but not BCL2L12A, whose C terminus lacks a binding region. We found that a BCL2L12153–191 fragment located outside of the C-terminal BH2 motif is responsible for GSK3b binding. In contrast, no interaction was detected between BCL2L12A and GSK3b. In vitro kinase and l-phosphatase assays showed that GSK3b phosphorylates BCL2L12 at S156, while this site is absent on BCL2L12A. Moreover, our data also showed that the BCL2L12153–191 fragment directly interrupted GSK3bmediated Tau phosphorylation in a dose-dependent manner. Ectopic expression of GFP-fused BCL2L12 or BCL2L12A in U87MG cells leads to repression of apoptotic markers and protects against staurosporine (STS) insults, indicating an antiapoptotic role for both BCL2L12 and BCL2L12A. In contrast, no anti-apoptotic ability was seen in BCL2L12(S156A). When BCL2L12-expressing U87MG cells were co-administrated with STS and LiCl, cells underwent apoptosis. This effect could be reversed by LiCl. In short, we established a model to demonstrate that GSK3b interacts with and phosphorylates BCL2L12 and might also affect BCL2L12A to modulate the apoptosis signaling pathway in glioblastoma. These findings suggest that LiCl may be a prospective therapeutic agent against GBM.