Comparative analysis of primate and pig cells reveals primate-specific PINK1 expression and phosphorylation

PTEN-induced putative kinase 1(PINK1),a mitochondrial kinase that phosphorylates Parkin and other proteins,plays a crucial role in mitophagy and protection against neurodegeneration.Mutations in PINK1 and Parkin can lead to loss of function and early onset Parkinson's disease.However,there is a lack of strong in vivo evidence in rodent models to support the theory that loss of PINK1 affects mitophagy and induces neurodegeneration.Additionally,PINK1 knockout pigs(Sus scrofa)do not appear to exhibit neurodegeneration.In our recent work involving non-human primates,we found that PINK1 is selectively expressed in primate brains,while absent in rodent brains.To extend this to other species,we used multiple antibodies to examine the expression of PINK1 in pig tissues.In contrast to tissues from cynomolgus monkeys(Macaca fascicularis),our data did not convincingly demonstrate detectable PINK1expression in pig tissues.Knockdown of PINK1 in cultured pig cells did not result in altered Parkin and BAD phosphorylation,as observed in cultured monkey cells.A comparison of monkey and pig striatum revealed more PINK1-phosphorylated substrates in the monkey brain.Consistently,PINK1 knockout in pigs did not lead to obvious changes in the phosphorylation of Parkin and BAD.These findings provide new evidence that PINK1expression is specific to primates,underscoring the importance of non-human primates in investigating PINK1function and pathology related to PINK1 deficiency.

LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization

Objective YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors;differentiating between these roles may depend on the YAP1 phosphorylation pattern.The specific function of YAP1 in B cell acute lymphoblastic leukemia(B-ALL),however,is currently unclear.Thus,in the present study,the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets.Methods The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting,quantitative real-time polymerase chain reaction,flow cytometry,immunostaining,and nude mouse subcutaneous tumorigenesis experiments.Gene expression levels of Hippo pathway-related molecules before and after verteporfin(VP)treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells.Results Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels.YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells;YAP1 was distributed in the nuclei in NALM6 cells.Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase.Before and after VP treatment,the expression of the upstream gene LATS1 was upregulated;its overexpression promoted YAP1-Ser127 phosphorylation.Further,YAP1 was distributed in the plasma.Conclusion LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function;thus,VP,which targets this axis,may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

5-氨基酮戊酸光动力治疗对鲍温病皮损中p53 Caveolin-1表达的影响

目的:探讨5-氨基酮戊酸光动力疗法对鲍温病皮损中p53、Caveolin-1表达的影响及意义。方法:运用5-氨基酮戊酸光动力疗法治疗鲍温病及周围正常皮肤组织各40例,采用免疫组织化学技术(SP法)检测光动力治疗前后鲍温病及周围正常皮肤组织中p53、Caveolin-1的阳性细胞表达率。结果:治疗前p53蛋白在正常皮肤组织及BD中表达的阳性率分别为10%、40%(χ^(2)=11.202,P<0.001),差异有统计学意义。治疗后p53在正常皮肤组织及BD的阳性率为10%、5%(χ^(2)=0.712,P=0.399),两者间差异无统计学意义。治疗前后p53在BD中的阳性表达率差异有统计学意义(χ^(2)=14.811,P<0.001)。治疗前Caveolin-1在正常皮肤组织及BD中阳性表达率为15%、55%(χ^(2)=14.449,P<0.001),两者间差异有统计学意义。治疗后正常皮肤组织及BD中Caveolin-1的阳性率为5%、12.5%(χ^(2)=0.816,P=0.366),差异无统计学意义。治疗前后Caveolin-1在BD中的阳性表达率差异有统计学意义(χ^(2)=19.013,P<0.001)。BD中p53与Caveolin-1阳性表达呈正相关性(r=0.533,P=0.015)。结论:p53及Caveolin-1的高表达可能与BD的发生密切关联,且ALA-PDT能够抑制p53及Caveolin-1的表达,从而抑制疾病的发展。通过更大样本量的研究,p53及Caveolin-1可能会成为皮肤相关疾病的诊断工具,并为其治疗提供新的靶点。

Caveolin-1介导流体剪切应力调控MC3T3-E1成骨细胞增殖和凋亡

背景:流体剪切应力在成骨细胞增殖和凋亡中起着重要作用。然而,Caveolin-1(Cav-1)是否参与成骨细胞中流体剪切应力诱导的增殖与凋亡过程尚不清楚。目的:探讨Cav-1在流体剪切应力调控成骨细胞增殖和凋亡中的作用。方法:选择生长状态良好的MC3T3-E1成骨细胞,加载不同时间(0,30,60,90 min)且强度为1.2 Pa的流体剪切应力,观察Cav-1蛋白的表达,筛选出时间为60 min的条件进行实验。将MC3T3-E1细胞分为:对照组、流体剪切应力组、流体剪切应力+pcDNA 3.1组(对照)、流体剪切应力+pcDNA Cav-1组(过表达质粒),分别采用流体剪切应力和过表达Cav-1等方式干预,通过q RT-PCR和Western blot检测MC3T3-E1细胞内增殖以及凋亡相关分子的表达量;通过CCK-8与Ed U实验检测MC3T3-E1细胞增殖活性;采用Hoechst 33258染色以及流式细胞术检测MC3T3-E1细胞凋亡情况。结果与结论:加载流体剪切应力后MC3T3-E1细胞中Cav-1的表达显著下调,且加载60 min表达水平最低。过表达Cav-1减弱了流体剪切应力促进MC3T3-E1细胞增殖及抑制细胞凋亡的效应。说明Cav-1在流体剪切应力调节成骨细胞增殖和凋亡过程中具有重要的作用,并可能为骨质疏松症提供潜在治疗策略。

四逆汤饼灸对兔膝骨关节炎软骨中Caveolin-1/p38 MAPK信号通路蛋白表达的影响

目的探讨四逆汤饼灸治疗膝骨关节炎(KOA)的作用机制。方法取新西兰兔50只,随机分为对照组、模型组、p38 MAPK抑制剂(抑制剂)组、四逆汤饼灸组、四逆汤饼灸联合p38 MAPK抑制剂(联合)组,每组10只。除对照组外,其他组采用右后肢膝关节伸直位石膏管型固定制备兔KOA模型。对照组、模型组不予治疗;抑制剂组于关节腔内注射p38 MAPK抑制剂TAK-715,1次/d,治疗2周;四逆汤饼灸组取鹤顶、内外膝眼穴位予以四逆汤饼灸治疗,1次/d,治疗2周;联合组于关节腔内注射p38 MAPK抑制剂TAK-715,并在鹤顶、内外膝眼穴位予以四逆汤饼灸治疗,1次/d,治疗2周。采用HE染色观察软骨病理变化,免疫组织化学染色法和蛋白质印迹法(Western blot)检测软骨组织中Caveolin-1、p38 MAPK、MMP-13蛋白的表达。结果HE染色结果显示,模型组软骨表面明显粗糙,破坏严重,细胞排列紊乱,Mankin’s评分明显升高;抑制剂组、四逆汤饼灸组及联合组软骨表面较光滑,软骨细胞分布较均匀,Mankin’s评分降低。免疫组化与Western blot结果显示,与对照组比较,模型组软骨组织中Caveolin-1、p38 MAPK、MMP-13的表达均升高(P均<0.05);与模型组比较,抑制剂组、四逆汤饼灸组及联合组软骨组织中Caveolin-1、p38MAPK、MMP-13的表达均降低(P均<0.05)。结论四逆汤饼灸能延缓KOA软骨的进一步破坏,其机制可能与抑制Caveolin-1/p38 MAPK信号通路蛋白的表达有关。

Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Our recent study shows that NEK2A interacts with MAD 1 at the kinetochore and possibly functions as a novel integrator of spindle checkpoint signaling. However, it is unclear how NEK2A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates human Sgo 1 and such phosphorylation is essential for faithful chromosome congression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore of mitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgo 1 is a substrate of NEK2A and the phosphorylation sites were mapped to Ser^14 and Ser^507 as judged by the incorporation of 32^P. Although such phosphorylation is not required for assembly of HsSgo 1 to the kinetochore, expression of non-phosphorylatable mutant HsSgo 1 perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation ofHsSgo 1 in orchestrating dynamic kinetochore-microtubule interaction. We propose that NEK2A-mediated phosphorylation of human Sgo 1 provides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.

Radiomics signature:A potential biomarker forβ-arrestin1 phosphorylation prediction in hepatocellular carcinoma

BACKGROUND The phosphorylation status ofβ-arrestin1 influences its function as a signal strongly related to sorafenib resistance.This retrospective study aimed to develop and validate radiomics-based models for predictingβ-arrestin1 phosphorylation in hepatocellular carcinoma(HCC)using whole-lesion radiomics and visual imaging features on preoperative contrast-enhanced computed tomography(CT)images.AIM To develop and validate radiomics-based models for predictingβ-arrestin1 phosphorylation in HCC using radiomics with contrast-enhanced CT.METHODS Ninety-nine HCC patients(training cohort:n=69;validation cohort:n=30)receiving systemic sorafenib treatment after surgery were enrolled in this retrospective study.Three-dimensional whole-lesion regions of interest were manually delineated along the tumor margins on portal venous CT images.Radiomics features were generated and selected to build a radiomics score using logistic regression analysis.Imaging features were evaluated by two radiologists independently.All these features were combined to establish clinico-radiological(CR)and clinico-radiological-radiomics(CRR)models by using multivariable logistic regression analysis.The diagnostic performance and clinical usefulness of the models were measured by receiver operating characteristic and decision curves,and the area under the curve(AUC)was determined.Their association with prognosis was evaluated using the Kaplan-Meier method.RESULTS Four radiomics features were selected to construct the radiomics score.In the multivariate analysis,alanine aminotransferase level,tumor size and tumor margin on portal venous phase images were found to be significant independent factors for predictingβ-arrestin1 phosphorylation-positive HCC and were included in the CR model.The CRR model integrating the radiomics score with clinico-radiological risk factors showed better discriminative performance(AUC=0.898,95%CI,0.820 to 0.977)than the CR model(AUC=0.794,95%CI,0.686 to 0.901;P=0.011),with increased clinical usefulness confirmed in both the training and validation cohorts using decision curve analysis.The risk ofβ-arrestin1 phosphorylation predicted by the CRR model was significantly associated with overall survival in the training and validation cohorts(log-rank test,P<0.05).CONCLUSION The radiomics signature is a reliable tool for evaluatingβ-arrestin1 phosphorylation which has prognostic significance for HCC patients,providing the potential to better identify patients who would benefit from sorafenib treatment.

Mitotic phosphorylation of PRC1 at Thr470 is required for PRC1 oligomerization and proper central spindle organization

During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with the centromere. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution ofcytokinesis. Central spindle organization requires mitotic kinesins, the chromosomal passenger protein complex, and microtubule bundling protein PRC 1. PRC 1 is phosphorylated by Cdc2 at Thr470 and Thr481 during mitosis. However, the functional relevance of PRC 1 phosphorylation at Thr470 has remained elusive. Here we show that expression of the non-phosphorylatable mutant PRC 1T470A but not the phospho-mimicking mutant PRC 1^T470E causes aberrant organization of the central spindle. Immunoprecipitation experiment indicates that both PRC 1^T470A and PRC 1^T470E mutant proteins associate with wild-type PRC 1, suggesting that phosphorylation of Thr470 does not alter PRC 1 self-association. In addition, in vitro co-sedimentation experiment showed that PRC 1 binds to microtubule independent of the phosphorylation state of Thr470. Gel-filtration experiment suggested that phosphorylation of Thr470 promotes oligomerization of PRC 1. Given the fact that prevention of the Thr470 phosphorylation inhibits PRC 1 oligomerization in vitro and causes an aberrant organization of central spindle in vivo, we propose that this phosphorylation-dependent PRC 1 oligomerization ensures that central spindle assembly occurs at the appropriate time in the cell cycle.

PTEN-induced kinase 1-induced dynamin-related protein 1 Ser637 phosphorylation reduces mitochondrial fission and protects against intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R)occurs in various diseases,such as trauma and intestinal transplantation.Excessive reactive oxygen species(ROS)accumulation and subsequent apoptotic cell death in intestinal epithelia are important causes of I/R injury.PTEN-induced putative kinase 1(PINK1)and phosphorylation of dynamin-related protein 1(DRP1)are critical regulators of ROS and apoptosis.However,the correlation of PINK1 and DRP1 and their function in intestinal I/R injury have not been investigated.Thus,examining the PINK1/DRP1 pathway may help to identify a protective strategy and improve the patient prognosis.AIM To clarify the mechanism of the PINK1/DRP1 pathway in intestinal I/R injury.METHODS Male C57BL/6 mice were used to generate an intestinal I/R model via superior mesenteric artery occlusion followed by reperfusion.Chiu’s score was used to evaluate intestinal mucosa damage.The mitochondrial fission inhibitor mdivi-1 was administered by intraperitoneal injection.Caco-2 cells were incubated in vitro in hypoxia/reoxygenation conditions.Small interfering RNAs and overexpression plasmids were transfected to regulate PINK1 expression.The protein expression levels of PINK1,DRP1,p-DRP1 and cleaved caspase 3 were measured by Western blotting.Cell viability was evaluated using a Cell Counting Kit-8 assay and cell apoptosis was analyzed by TUNEL staining.Mitochondrial fission and ROS were tested by MitoTracker and MitoSOX respectively.RESULTS Intestinal I/R and Caco-2 cell hypoxia/reoxygenation decreased the expression of PINK1 and p-DRP1 Ser637.Pretreatment with mdivi-1 inhibited mitochondrial fission,ROS generation,and apoptosis and ameliorated cell injury in intestinal I/R.Upon PINK1 knockdown or overexpression in vitro,we found that p-DRP1 Ser637 expression and DRP1 recruitment to the mitochondria were associated with PINK1.Furthermore,we verified the physical combination of PINK1 and p-DRP1 Ser637.CONCLUSION PINK1 is correlated with mitochondrial fission and apoptosis by regulating DRP1 phosphorylation in intestinal I/R.These results suggest that the PINK1/DRP1 pathway is involved in intestinal I/R injury,and provide a new approach for prevention and treatment.

Hepatitis C Virus non-structural 5A abrogates signal transducer and activator of transcription-1 nuclear translocation induced by IFN-α through dephosphorylation

AIM： To study the effect of Hepatitis C virus nonstructural 5A （HCV NSSA） on IFNα induced signal transducer and activator of transcription-1 （STAT1） phosphorylation and nuclear translocation.METHODS： Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NSSA-expressed and non-HCV NSSA-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells.RESULTS： Expression of HCV NS5A was found in the cytoplasm of pCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells, STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction, STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to pRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot.CONCLUSION： HCV NSSA expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells

AIM： To study the relationship between interleukin-lbeta （IL-1β） up-regulating tissue inhibitor of matrix metalloproteinase-1 （TIMMP-1） mRNA expression and phosphorylation of both c-jun N-terminal kinase （INK） and p38 in rat heffatic stellate cells （HSC）. METHODS： RT-PCR was performed to measure the expression of TIMMP-1 mRNA in rat HSC. Western blot was performed to measure IL-1β-induced JNK and p38 activities in rat HSC. RESULTS： TIMMP-1 mRNA expression （1.191± 0.079） was much higher after treatment with IL-1β （10 ng/mL） for 24 h than in control group （0.545±0.091） （P〈0.01）. IL-1β activated INK and p38 in a time-dependent manner. After stimulation with IL-1β for 0, 5, 15, 30, 60 and 120 min, the INK activity was 0.982±0.299, 1.501±0.720, 2.133±0.882, 3.360±0.452, 2.181±0.789, and 1.385 ± 0.368, respectively. There was a significant difference in JNK activity at 15 min （P〈 0.01）, 30 min （P〈 0.01） and 60 min （P〈0.01） in comparison to that at 0 min. The p38 activity was 1.061±0.310, 2.050±0.863, 2.380±0.573, 2.973±0.953, 2.421±0.793, and 1.755 ± 0.433 at the 6 time points （0, 5, 15, 30, 60 and 120 min） respectively. There was a significant difference in p38 activity at 5 min （P〈0.05）, 15 min （P〈0.01）, 30 min （P〈0.01） and 60 min （P〈0.01） compared to that at 0 min. TIMMP-1 mRNA expression trended to decrease in 3 groups pretreated with different concentrations of SP600125 （10 μmol/L, 1.022±0.113; 20 μmol/L, 0.869±0.070; 40 μmol/L, 0.666±0.123）. Their decreases were all significant （P〈0.05, P〈0.01, P〈0.01） in comparison to control group （without SP600125 treatment, 1.163±0.107）. In the other 3 groups pretreated with different concentrations of SB203580 （10 μmol/L, 1.507±0.099; 20 μmol/L, 1.698±0.107; 40 μmol/L, 1.857±0.054）, the expression of TIMMP-1 mRNA increased. Their levels were higher than those in the control group （without SB203580 treatment, 1.027 ± 0.061） with a significant statistical significance （P〈 0.01）. CONCLUSION： IL-1β has a direct action on hepatic fibrosis by up-regulating TIMMP-1 mRNA expression in ratessionin in rate HSC.JNK and p38 mitogen-activated protein kinases （MAPKs） are involved in IL-1β-induced TIMMP-1 gene expression, and play a distinct role in this process, indicating that p38 and .INK pathways cooperatively mediate TIMP-1 mRNA expression in rat HSC.

Caveolin-1与疾病发生相关性的研究进展

Caveolin-1(CAV1)是一种致癌膜蛋白,与胞吞作用、细胞外基质组织、胆固醇分布、细胞迁移和信号传导有关,在多种疾病中改变的CAV1通过控制新陈代谢来影响疾病的发展或进展。本文主要介绍了Caveolin-1在癌症、糖尿病、其他生理疾病中的作用以及在疾病发生发展方面的研究现状。

Phosphorylation of AMMECR1 at Serine16 Is Not Essential for Its Nuclear Localization

AMMECR1 is a critical gene in the pathogenesis of AMME syndrome. But, little is known about how AMMECR1 is regulated. Here, we showed that many human cell lines expressed AMMECR1. With Phos-tag SDS PAGE analysis, we demonstrated that AMMECR1 was constitutively phosphorylated at Ser16. AMMECR1 was localized in the nucleus. Mutation of Ser16 to alanine did not affect its nuclear localization. The homologs of AMMECR1, PAC688.03c and MTH857, were also nuclear proteins, but they were not phosphorylated when tested in HeLa cells. Therefore, AMMECR1 and its homologs might have atypical nuclear localization sequences.

Role of Notoginsenoside Rg1 in Improving Spatial Cognitive Ability and Lowering Phosphorylation Level of Tau Protein in AD Model Rats

[Objectives] To study the effects and mechanism of notoginsenoside Rg1 on the spatial learning and memory and phosphorylated tau protein in the AD( Alzheimer's Disease) model rat. [Methods]The AD model rat was replicated by injection of Aβ_(25-35) in the left lateral ventricles of SD rats. The low dose( 25 mg/kg),middle dose( 50 mg/kg) and high dose( 100 mg/kg) notoginsenoside Rg1 was used for intragastric administration,respectively,two times every day. After 4 weeks,the Morris water maze test was done to detect the learning and memory capacity,and the immunoblotting,immunohistochemical methods were used to detect the changes in the phosphorylation level and distribution of tau protein in hippocampus of the rats. [Results] After the intracerebroventricular injection of Aβ_(25-35),the learning and memory capacity of the model rats was significantly lower than the learning and memory capacity of the normal control rats. The immunoblotting test results showed that the phosphorylation level of tau protein threonine 231 site( Thr231) in hippocampus was significantly increased,and the nonphosphorylation level was significantly decreased. The morphological testing results showed that the phosphorylation level of tau protein Thr231 of AD model rats was increased markedly in region of DG,CA1 and CA3 of the hippocampus. The intervention of the middle dose notoginsenoside Rg1 could significantly improve the learning and memory capacity of the model rats in Morris water maze. The notoginsenoside Rg1 in three different doses could all reduce the phosphorylation level of tau protein Thr231 in the hippocampal DG,CA1,CA3 regions,and there were no significant differences among the three doses. [Conclusions]The notoginsenoside Rg1 could improve Aβ_(25-35)-induced spatial learning and memory impairment of the AD model rats,and decreased the phosphorylation level of tau protein in hippocampus.

Correlations of the expression of Cx43,SCFFBXW7,p-cyclin E1(Ser73),p-cyclin E1(Thr77)and p-cyclin E1(Thr395)in colon cancer tissues

BACKGROUND Previous cellular studies have demonstrated that elevated expression of Cx43 promotes the degradation of cyclin E1 and inhibits cell proliferation through ubiquitination.Conversely,reduced expression results in a loss of this capacity to facilitate cyclin E degradation.The ubiquitination and degradation of cyclin E1 may be associated with phosphorylation at specific sites on the protein,with Cx43 potentially enhancing this process by facilitating the phosphorylation of these critical residues.AIM To investigate the correlation between expression of Cx43,SKP1/Cullin1/F-box(SCF)FBXW7,p-cyclin E1(ser73,thr77,thr395)and clinicopathological indexes in colon cancer.METHODS Expression levels of Cx43,SCFFBXW7,p-cyclin E1(ser73,thr77,thr395)in 38 clinical colon cancer samples were detected by immunohistochemistry and were analyzed by statistical methods to discuss their correlations.RESULTS Positive rate of Cx43,SCFFBXW7,p-cyclin E1(Ser73),p-cyclin E1(Thr77)and p-cyclin E1(Thr395)in detected samples were 76.32%,76.32%,65.79%,5.26%and 55.26%respectively.Positive expressions of these proteins were not related to the tissue type,degree of tissue differentiation or lymph node metastasis.Cx43 and SCFFBXW7(r=0.749),p-cyclin E1(Ser73)(r=0.667)and p-cyclin E1(Thr395)(r=0.457),SCFFBXW7 and p-cyclin E1(Ser73)(r=0.703)and p-cyclin E1(Thr395)(0.415)were correlated in colon cancer(P<0.05),and expressions of the above proteins were positively correlated in colon cancer.CONCLUSION Cx43 may facilitate the phosphorylation of cyclin E1 at the Ser73 and Thr195 sites through its interaction with SCFFBXW7,thereby influencing the ubiquitination and degradation of cyclin E1.

Mec1-Dependent Phosphorylation of the Scc3 Subunit of Cohesin during Mitosis in Budding Yeast

Cohesin is an evolutionary conserved complex that controls chromosome segregation during mitosis. Here we show that, in response to DNA damage, Saccharomyces cerevisiae Scc3, one of the major regulatory subunits of the Smc1-Smc3-Scc1 cohesin ring, is phosphorylated on S/T-Q residues. This event depended on the Mec1 checkpoint kinase as well as on cell cycle arrest triggered by the DNA damage checkpoint network. This phosphorylation event also took place during mitosis of an unperturbed cell cycle. The present finding that S. cerevisiae Scc3 is phosphorylated during mitosis represents a potentially important new regulatory step in cohesin’s mitotic functions.

Phosphorylation on TRPV4 Serine Residue 824 Enhances Its Association with PGM1

The TRPV4 cation channel is expressed in a broad range of tissues and participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, human phosphoglucomutase- 1 (PGM1), an enzyme that converts glucose-6 phosphate to glucose-1 phosphate in the glycolysis pathway, as the first auxiliary protein of TRPV4 Ca2+ channels, is identified with yeast two hybrid system, coimmunoprecipitation, confocal microscopy, and GST pull-down assays. TRPV4 forms a complex with PGM1 through its C-terminal cytoplasmic domain. Because it is demonstrated that TRPV4 serine residue 824 (S824) is phosphorylated by serum/glucocorticoid regulated kinase 1, we elucidate the effect of TRPV4 S824 phosphorylation on TRPV association with PGM1. Even an inactivated mutant version of TRPV4, S824A, exhibited a decreased ability to bind PGM1, an activated phosphomimetic mutant version of TRPV4, S824D, exhibited enhanced binding to PGM1. Thus, formation of the TRPV4/PGM1 complex and localization of this complex to the plasma membrane appear to be regulated by the phosphorylation status of residue S824 in TRPV4. The newly identified interactor of TRPV4 may help the molecular pathways modulating transport activity or glucose metabolism, respectively.

Regulation of Breast Cancer Progression by Phosphorylation of the Tumor Suppressor Tropomyosin-1 Alpha

Background: Tropomyosin 1 alpha chain (Tm1) is an actin-binding protein that regulates the endothelial cell response to oxidative stress following its phosphorylation at Serine 283 (S283). Tm1 is also a major tumor suppressor in breast cancer. In the present study, we investigated the role of phosphorylation of Tm1 in regulating its tumor suppressor properties. Methods: MDA MB231 breast cancer cells stably overexpressing wild type form of Tm1 or Tm1 mutants (S283A and S283E) were generated. Proliferation and cell viability were assayed by means of the enzymatic cleavage of the tetrazolium salt WST-1 to formazan dye by cellular mitochondrial dehydrogenases. Adhesion assays were performed at various periods of time on cells grown on plastic. Cell migration was evaluated by using the wound-healing assay and by measuring transendothelial migration of cancer cells. Malignant transformation in vitro was determined by using the anchorage-independent growth assay on soft agar. Results: We found that cells expressing the phosphomimetic form of Tm1 S283E/Tm1 are characterized by an increased adhesion to the substratum. Moreover, the migration of MDA-MB231/S283E/Tm1 cells in a wound closure assay is reduced compared to parental cells or those expressing the non-phosphorylatable form of Tm1 (S283A). Similarly, the transendothelial migration of MDA-MB231/S283E/Tm1 cells is also reduced as compared to the other cell lines. Moreover, we found that the cells expressing the S283A mutants form more colonies in soft agar that those expressing the S283E mutants. Conclusion: Phosphorylation of Tm1 at Ser283 contributes to its anti-tumor properties, and this effect results mainly from an increase in cell adhesion associated with a decrease in their migratory and invasive potentials.

大鼠脑内caveolin-1蛋白的表达及其在分辨学习中的作用

Caveolin-1(Cav-1)蛋白作为细胞质膜结构小窝(caveolae)的标志蛋白,在胆固醇运输、膜组装、信号转导和细胞转化过程中扮演重要的角色。为了探讨Cav-1蛋白在中枢神经系统可塑性及学习记忆中的作用,本文以Sprague-Dawley大鼠为实验对象,利用蛋白质免疫印迹杂交方法观察了Cav-1蛋白在不同年龄大鼠脑内表达的特征,并研究了Y-迷宫训练前后Cav-1蛋白表达的变化。结果表明:(1)大鼠不同脑区Cav-1蛋白表达的年龄特征不同。海马内的表达属青年鼠最高,其次是老年鼠和幼年鼠;皮层内的表达属幼年鼠最高,其次是老年鼠,青年鼠最低;小脑内的表达无明显年龄差异。(2)Y-迷宫训练引起青年鼠海马和前额叶皮层内Cav-1蛋白的表达显著增加。结果提示,Cav-1蛋白与动物脑发育和学习记忆有密切关系,可能参与中枢可塑性的调节。

Caveolin-1、GPR30和Vimentin在甲状腺乳头状癌组织中的表达及意义

目的:探讨Caveolin-1、GPR30和Vimentin在人甲状腺乳头状癌(Papillary thyroid carcinoma,PTC)的表达及意义。方法:选取甲状腺乳头状癌76例和正常的甲状腺组织44例,采用免疫组织化学S-P法检测Caveolin-1、GPR30和Vimentin的表达,分析其与患者临床病理指标的关系,以及三者之间表达的相关性。结果:在PTC组织中,Caveolin-1、GPR30和Vimentin的阳性表达率分别为9.21%(7/76)、80.26%(61/76)、76.32%(58/76),其中Caveolin-1的阳性表达率明显低于正常组织81.82%(36/44),而GPR30、Vimentin的阳性表达率明显高于正常组织0(0/44)、0(0/44),差异具有统计学意义(P<0.001)。Caveolin-1、GPR30和Vimentin的表达与肿瘤的分期(P=0.005,P<0.001,P<0.001)以及颈部淋巴结转移(P≤0.001)显著相关。此外Caveolin-1与GPR30的表达呈负相关(rs=-0.528,P<0.001),Caveolin-1与Vimentin的表达呈负相关(rs=-0.572,P<0.001),GPR30与Vimentin的表达呈正相关(rs=0.812,P<0.001)。Caveolin-1联合GPR30或Vimentin表达的条件下:Caveolin-1阴性表达伴随GPR30或Vimentin阳性表达与颈部淋巴结转移更具有相关性(P<0.05);GPR30与Vimentin联合阳性表达较只有一种蛋白阳性表达与颈部淋巴结转移更具有相关性(P=0.005);Caveolin-1阴性表达伴随GPR30和Vimentin阳性表达与颈部淋巴结转移的相关性最强(P<0.001)。结论:Caveolin-1、GPR30和Vimentin在PTC组织中的表达与肿瘤分期,以及颈部淋巴结转移密切相关,且三者的表达密切相关,Caveolin-1、GPR30和Vimentin在PTC中的表达情况可作为检测PTC颈部淋巴结转移的指标。