Дисертації з теми "RhoGDIα"

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Les petites proteines g de la superfamille ras se comportent dans la cellule comme de veritables commutateurs moleculaires en oscillant entre une conformation inactive liee au gdp, et une conformation active liee au gtp. C'est l'integration d'un stimulus extracellulaire activateur qui permet le passage d'un etat a l'autre pour la transduction du signal. L'activation a une duree determinee dans le temps et dans l'espace cellulaire. L'orchestration des differentes interactions qui ont lieu entre les petites proteines g et leurs regulateurs est absolument cruciale pour mettre en place une reponse cellulaire specifique. Dans le cas des proteines de la famille rho, une autre famille de proteines regulatrices intervient en plus des gef et des gap : les proteines rhogdi. Les rhogdi regulent le cycle gtp/gdp et le cycle d'association/dissociation des proteines rho aux membranes. L'activation de la proteine rho implique que la stimulation de l'echange nucleotidique par le gef soit coordonnee a la dissociation de la proteine rhogdi. L'articulation de ces deux evenements est tres mal connue. Dans cette optique, l'etude de la proteine rhogdi-3 a ete entreprise. Nous avons montre que la proteine rhogdi-3 est une gdi non conventionnelle qui se distingue de ses deux isoformes par une association aux membranes de l'appareil de golgi. L'extremite n-terminale hydrophobe de la proteine rhogdi-3 est necessaire et suffisante pour cette localisation. De plus, rhogdi-3 regule negativement et specifiquement l'activite de la proteine rhog dans les cellules hela. Il a aussi ete mis en evidence l'existence d'un complexe cytoplasmique entre ces deux proteines et la capacite de rhogdi-3 a extraire rhog des membranes pour former un pool de complexes inactifs. L'ensemble de ces resultats nous conduit a faire l'hypothese que la proteine rhogdi-3 pourrait participer au recyclage de la proteine rhog inactive de la membrane plasmique vers l'appareil de golgi ou elle serait activee par le facteur d'echange trio.

碩士
國立陽明大學
生物藥學研究所
103
Megakaryocytes (MK) are derived from bipotent megakaryocyte/erythroid progenitors (MEP) of the myeloid lineage and are the sole source of circulating platelets which play a vital role in stopping bleeding. K562 is a human bipotent myeloleukemia cell line and has been used as a cell model for studying MK differentiation. Previous studies in this lab have shown that the p38α MAPK pathway negatively regulates MK differentiation. The molecular events upstream of p38α are yet to be elucidated. 　　Members of small GTPase Rho family are known to act upstream of MAPK pathways. The Rho GDP-dissociation factor α (RhoGDIα) acts to inhibit the activity of Rho GTPases. Our previous results showed that overexpression of RhoGDIα stimulated and knockdown of RhoGDIα suppressed PMA-induced MK differentiation. My results showed that RhoGDIα-overexpressing stable clones suppressed p38 activation, which is in agreement with our previous observation that knockdown of RhoGDIα enhanced p38 activation confirming that RhoGDIα negatively regulated p38 activation. It was reported that Arg 111, 152 and 180 of RhoGDIα are methylated. Our previous results showed that the R111/152/180K triple mutant failed to promote MK differentiation. My study with R111/152/180-expressing stable clones showed that not only MK was suppressed but also p38 activation was increased suggesting Arg 111/152/180 played a role in regulating p38 activation and MK differentiation. 　　I further investigated the potential downstream target of RhoGDIα. By ectopic expression, I showed that the constitutively active Rac1 not only suppressed differentiation but also displayed a significant dominant-negative effect whereas the constitutively active forms of RhoA did not affect differentiation. These results suggest that RhoGDIα may act through suppressing the activity of Rac1.

碩士
輔英科技大學
醫事技術系碩士班
97
In Taiwan, hepatocellular carcinoma ranks as the first and the second leading causes of death for male and female cancer patients, respectively. Recently, inappropriate activation of Wnt signaling pathway has been implicated in the development of HCC. Therefore, this study focused on searching for TCF-4 and beta-catenin regulated proteins. TCF-4 and β-catenin were transfected in Huh7. Our study found that TCF-4 and β-catenin down-regulated RhoGDIα expression in HCC by proteomic method. Furthermore, the levels of RhoGDIα expression in HCC tissue and peritumorous non-neoplastic liver tissue were determined by RT-PCR. The results indicated that RhoGDIα expression is down-regulated in human HCC compared with peritumorous non-neoplastic liver tissues. The functional analysis of RhoGDIα in tumor cell was defined further by apoptosis assay, MTT assay, migration and invasion assay. Ectopic expression of RhoGDIα altered tumor cell survival, apoptosis and migration. The results suggest that down-regulation of RhoGDIα expression plays a significant role in the progress of hepatocellular carcinoma.

碩士
國立陽明大學
生物藥學研究所
101
Megakaryocytes are the precursors of platelets and thus play an essential role in blood clotting. K562 is a human leukemia cell line that can be induced by PMA (phorbol 12-myristate 13-acetate) to undergo megakaryocytic (MK) differentiation and has been used as a cell model for studying MK differentiation. The Rho family belongs to the small GTPase superfamily and GDP-dissociation inhibitor α (RhoGDIα) suppresses the activity of Rho. Our previous study has shown that RhoGDIα promoted MK differentiation. RhoGDIα has been reported to contain three di-methylated arginines (R111, R152 and R180) however their function is not reported. In addition, our previous results also showed that protein arginine methyltransferase 6 (PRMT6) played a positive role in MK differentiation. This study thus aimed to investigate the functional role of arginine methylation of RhoGDIα and whether PRMT6 mediates methylation of RhoGDIα. In this study, the arginine residues (R111, R152 and R180) were mutated to lysines to mimic the non-methylated state. By ectopic expression, my results showed that single, double and triple mutations lost, to different extent, their stimulatory effects on MK differentiation of K562 cells. Besides R180K, all the mutants appeared to have a dominant negative effect. In the RhoGDIα knockdown (KD) cells, R111K and R152K single mutants still promoted MK differentiation however to a lower degree than wild type did. R111/152K and R111/152/180K double mutants completely lost their stimulatory effects. R111/180K and R152/180K had a similar effect to R111K and R152K single mutants. Together, these results suggest methylation on R111 and R152 plays a more significant role than R180 in promoting MK differentiation. Overexpression of PRMT6 in RhoGDIα KD cells could no longer promoted MK differentiation. When co-expressed with RhoGDIα, PRMT6 regains its ability to promote differentiation in RhoGDIa knockdown cell; while co-expression with RhoGDIα R111/152K did not help regaining the ability. Notably, PRMT6 methylated RhoGDIα in in vitro methylation. These results suggest that PRMT6 may be responsible for methylation of R111 and R152 of RhoGDIα which then promote PMA-induced megakaryocytic differentiation of K562 cells. This study provides links suggesting a positive role of RhoGDIα arginine methylation in MK differentiation.

碩士
國立陽明大學
生物藥學研究所
99
The mitogen-activated protein kinase (MAPK) pathways regulate various cellular functions, including differentiation. Activation of Erk MAPK pathway promotes megakaryocytic (MK) differentiation and p38 MAPK pathway plays a negative role in MK differentiation. K562 is a multipotent human leukemia cell line which can be induced to differentiate into various lineages including megakaryocytes. Upon PMA (phorbol 12-myristate 13-acetate) treatment, K562 cells display characteristics of MK and are often used as a model cell line. Our previous study demonstrated that PRMT1 (protein arginine methyltransferase 1) inhibited PMA-induced MK differentiation of K562 cells via activation of p38? MAPK. However, the molecular mechanism is unclear. The activity of the p38 MAPK pathway is regulated by the upstream MAPKK and MAPKKK and small G proteins such as Rho family. The Rho GDP-dissociation factor ? (RhoGDI?? is known to inhibit activity of Rho GTPases and has been shown to contain three arginine residues that can be dimethylated. This study showed that RhoGDI? plays a positive role in PMA-induced MK differentiation of K562 cells as demonstrated by changes in cytological characteristics. Activation of p38 was enhanced in RhoGDI? knockdown cells by Western blot analysis. Suppression of MK differentiation in RhoGDI? knockdown cells was reverted upon treatment of p38 inhibitor (SB203580). RhoGDI? could reverse the MK differentiation in p38β knockdown cells but not in p38? knockdown cells. In context with PRMT1, promotion of MK differentiation by RhoGDI? was reversed. Mutations at Arg111 or Arg152, which are known to be methylated in cells, abolished the promotion of MK differentiation by RhoGDI?? Together, our results suggest, for the first time, that RhoGDI? and the potential role of methylation regulates PMA-induced megakaryocytic differentiation of K562.

碩士
中山醫學大學
口腔醫學研究所
92
The leucine-zipper (LZ) and sterile- motif (SAM) kinase (ZAK) belongs to the MAP kinase kinase kinase (MAP3K) when upon over expression in mammalian cells activates the JNK/SAPK pathway, comprising a group of highly related serine / threonine kinase. That ZAK activates JNK / SAPK mediated by downstream taget, MKK7. The report present suggests a schematic pathway of ZAK→MKK7→JNK→cell arrest. By substrative yeast two hybrid system to cloned a RhoGDI- homologous cDNA, named RhoGDIβ. Rho GDP-dissociation inhibitors (RhoGDIs), endogenous inhibitors of Rho GTPase, play an important role in regulating the biological activitys of Rho protein. As yet, only three RhoGDIs have been described：RhoGDIα(RhoGDI, RhoGDI-1), RhoGDIβ(D4 / Ly-GDI, RhoGDI- 1), and RhoGDIγ(RhoGDI-3). We used GST-pull down to prove that the relationship between ZAK and RhoGDIβ. And by tet-on system suggest that RhoGDIβhave effect on some Cell cycle regulator protein. Besides, it also identifies RhoGDIβ regulat the small GTPase protein, Rac.

Muscle contractions and exercise have been shown to potently stimulate muscle glucose uptake via molecular mechanisms that are different to insulin-stimulated glucose uptake. Importantly, although insulin-stimulated glucose uptake is diminished in people with type 2 diabetes, the ability for contractions or exercise to stimulate muscle glucose uptake appears to be preserved. Activating this exercise signalling pathway with novel drugs could theoretically bypass the defective insulin-signalling pathway and lower blood glucose levels in insulin-resistant individuals. However, the exact signalling mechanisms involved require a better understanding. Several potential regulators of contraction-stimulated glucose uptake have been identified and some degree of redundancy probably exists. Nitric oxide (NO) has been suggested to regulate muscle glucose uptake in rodent and human models. People with type 2 diabetes appear to have a greater reliance on NO to regulate muscle glucose uptake during exercise compared to healthy individuals. Recently, Rac1 has been identified as a novel regulator of muscle glucose uptake during stretch, contraction, and exercise in rodent models. However, the exact mechanisms how both NO and Rac1 regulate glucose uptake are not fully understood. Interestingly, there is some evidence from cell culture studies suggesting an interaction between NO and Rac1 may exist, however, this remains untested in the context of contraction- or exercise-stimulated glucose up in mature muscle models. Therefore, in this thesis, I examined the regulation of skeletal muscle glucose uptake during stretch, contraction, and exercise with a focus on NOS and Rac1 signalling mechanisms. In particular, the potential for NOS and Rac1 to act via the same signalling pathway was explored. The pathways activated by stretch have been considered to overlap with contraction signalling. Stretch is known to increase glucose uptake via Rac1, and there is published evidence that stretch increase muscle NO levels. Therefore, in Study 1, an ex vivo muscle stretching model was used to examine whether NO also plays a role in stretch-stimulated glucose uptake. Stretch increased glucose uptake in isolated EDL muscles and treatment with the NO synthase (NOS) inhibitors L-NMMA and L-NMMA had no effect on this stretch- stimulated glucose uptake. Likewise, stretch-stimulated glucose uptake was normal in muscles from nNOSμ knockout (KO) and eNOS KO mice. A dissociation between NO and Rac1 was observed given that stretching stimulated an increase in Rac1 signalling but did not increase NOS activity above resting levels. This suggested that activation of Rac1 during stretch does not require NO to regulate glucose uptake. I was interested to further examine a potential NO and Rac1 interaction in skeletal muscle given there is evidence from previously published studies suggesting that increased levels of NO activate Rac1 in cells in culture. Therefore, in Study 2, I treated isolated muscles with a NO donor (DETA/NO) to increase NO levels, and this increased glucose uptake above resting levels. Addition of a Rac1 inhibitor (Rac1 inhibitor II) completely prevented this DETA/NO stimulation of skeletal muscle glucose uptake. This suggests that NO can stimulate glucose uptake via a pathway involving Rac1. However, when tested during muscle contraction to increase endogenous NO levels, NOS inhibition did not alter Rac1 signalling during muscle contraction. This suggests that in a more physiological setting, NO does not activate Rac1. Further evidence dissociating a NOS-Rac1 link is suggested by the observation that contraction-stimulated glucose uptake was attenuated by Rac1 inhibition but not by NOS inhibition. The finding that NOS inhibition did not attenuate glucose uptake was surprising as this contrasted with previous studies by our group. Therefore, these results provide further evidence that in regulating muscle glucose uptake, Rac1 signalling does not involve NO. Furthermore, these findings question the hypothesis that NOS plays an important role in the regulation of skeletal muscle glucose uptake during contraction. Given that findings from Study 1 and Study 2 suggest NOS does not play a role in regulating Rac1 signalling and skeletal muscle glucose uptake, I next turned attention to another promising candidate regulator of Rac1 signalling. RhoGDIα has previously been reported to negatively regulate Rac1 activity in cell culture models. It was important however to examine this in a more physiological model such as whole-body exercise where Rac1 is known to be a major regulator of skeletal muscle glucose uptake. Therefore, in Study 3, RhoGDIα was overexpressed in skeletal muscles of mice to test the hypothesis that exercise- stimulated glucose uptake would be attenuated by the negative action of RhoGDIα on Rac1 signalling. However, increased skeletal muscle RhoGDIα protein levels did not attenuate exercise-stimulated glucose uptake, and Rac1 signalling appeared to be normal. Interestingly, increased levels of Rac1 protein were observed in RhoGDIα overexpressing mice. Therefore, in contrast to previous cell culture studies where increased levels of RhoGDIα were found to reduce Rac1 signalling, such a negative role for RhoGDIα in a more physiological setting is not so clear. The increase in Rac1 protein levels in RhoGDIα overexpressing mice could have served to maintain Rac1 signalling during exercise and highlights the potential of a compensatory mechanism to protect signalling pathways regulating glucose metabolism. In summary, findings from this thesis show that during muscle stretching or contraction, NO and Rac1 are not linked. The most striking finding of this thesis is that we were unable to reproduce findings of previous work from our lab since we report that NOS inhibition does not attenuate contraction-stimulated glucose uptake. Given the observed dissociation between NOS and Rac1, another potential regulator of Rac1 signalling, RhoGDIα was examined. In contrast to previous cell culture studies, the role of RhoGDIα as a negative regulator of Rac1 in the context of exercise-stimulated glucose uptake is not clear, since RhoGDIα overexpression induced compensatory changes to other proteins including Rac1 that ultimately did not affect glucose uptake. Further work is required to unravel the complex regulation of Rac1 signalling towards glucose uptake in muscle.

博士
中山醫學大學
醫學研究所
98
ZAK belongs to the mixed lineage kinases, a family of serine/threonine kinases that are classified as MAP3Ks and can activate the JNK and nuclear factor κB (NFκB) pathway ZAK induces JNK activation through a dual phosphorylation kinase, JNKK2/MKK7. To identify effectors of ZAK and to study the ZAK signaling cascade,we used a yeast two-hybrid system to isolate ZAK effectors from a human heart cDNA library. One of the isolated cDNAs encoded Rho GDP dissociation inhibitor beta (RhoGDIβ).However, only their ability to sequester RhoGTPases is well understood. Therefore, the question remains as to whether RhoGDIβ is actually a RhoGTPase regulator. Overexpression of RhoGDIβ, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDIβ expression by RNA interference blocked hypertrophic growth. The further studies demonstrated that RhoGDIβ physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDIβ functions. Moreover, the ZAK-RhoGDIβ interaction may maintain ZAK in an inactive hypophosphorylated form. These two proteins could negatively regulate one another such that ZAK suppresses RhoGDIβ functions through phosphorylation and RhoGDIβ counteracts the effects of ZAK by physical interaction. Knockdown of ZAK expression in ZAK- and RhoGDIβ-expressing cells by ZAK-specific RNA interference restored the full functions of RhoGDIβ. At our previous study demonstrated that RhoGDIβ plays a undefined role in regulating Rac1 expression through transcription to induce hypertrophic growth and cell migration and that these functions are blocked by the expression of a dominant-negative form of Rac1.Knockdown of RhoGDIβ expression by RNA interference blocked RhoGDIβ-induced Rac1 expression and cell migration. We demonstrated that the co-expression of ZAK and RhoGDIβ in cells resulted in an inhibition in the activity of ZAK to induce ANF expression. Knockdown of ZAK expression in ZAK-RhoGDI β-expressing cells by ZAK-specific RNA interference restored the activities of RhoGDIβ. Expression of RhoGDIβ induces hypertrophic growth via modulation of Rac1 expression in H9c2 cardiac cells. H9c2 cell migration promoted by RhoGDIβ is Rac1 dependent . Knockdown of overexpressed RhoGDIβby siRNA reduces H9c2 cell migration. RhoGDIβ-induced cell migration does not correlate with cell proliferation ，and RhoGDIβ -induced wound healing is negatively regulated by ZAK.

碩士
中山醫學大學
口腔醫學研究所
96
To search effectors of ZAK and to study the ZAK signaling cascade, our laboratory used a yeast two-hybrid system to isolate ZAK associated proteins from a human heart cDNA library. One of the isolated cDNAs encoded Rho GDP dissociation inhibitor beta (RhoGDIβ). RhoGDIβ, also known as Ly-GDI or D4-GDI, which belongs to a family of Rho GDP dissociation inhibitors that includes RhoGDIα, RhoGDIβ and RhoGDIγ is thought to regulate many biological activities in cells and also regulate the activity and localization of Rho family proteins. RhoGDIβ is almost exclusively expressed in hematopoietic lineages. In the lab, we discovered that RhoGDIβ induced hypertrophic growth of a cultured rat cardiac cell line, H9c2. In order to study the mechanisms of RhoGDIβ to regulate hypertrophic growth, we used a yeast two-hybrid system to determine RhoGDIβ associated protein from a human heart cDNA library. Only four yeast colonies were growing in selection plates and we isolated these cDNAs. We then identified two possible RhoGDIβ associated proteins, RhoA and MYBPC3, after sequencing these cDANs. We set to determine the association of these proteins with RhoGDIβ in vivo in this study.

Shortcomings of current methods of prostate cancer detection draw attention to a need for improved biomarkers. The TMPRSS2:ERG gene fusion leads to the overexpression of ERG, an ETS family transcription factor, and is the most prevalent genetic lesion in prostate cancer, but its clinical utility remains to be defined. Two radical prostatectomy samples were analysed by next-generation whole transcriptome sequencing. The chosen samples differed in fusion gene status, as previously determined by RT-PCR. The involvement of novel and previously reported prostate cancer-related transcripts, Wnt signalling, p53 effector loss and several ETS-regulated pathways was identified in the prostate cancer cases examined. ERG was found to directly transactivate RhoGDIB, a gene associated with fusion-positive prostate cancer. Overexpression of RhoGDIB elicited spindle-shaped morphology, faster cell migration and increased cell proliferation, phenotypic changes suggestive of cancer progression. The present findings confirm the value of comprehensive sequencing for biomarker development and indicate avenues of future study.