BACKGROUND Cancerous inhibitor of protein phosphatase 2A(CIP2A)is a newly discovered oncogene.It is an active cell proliferation regulatory factor that inhibits tumor apoptosis in gastric cancer(GC)cells.CIP2A is func...BACKGROUND Cancerous inhibitor of protein phosphatase 2A(CIP2A)is a newly discovered oncogene.It is an active cell proliferation regulatory factor that inhibits tumor apoptosis in gastric cancer(GC)cells.CIP2A is functionally related to chemoresistance in various types of tumors according to recent studies.The underlying mechanism,however,is unknown.Further,the primary treatment regimen for GC is oxaliplatin-based chemotherapy.Nonetheless,it often fails due to chemoresistance of GC cells to oxaliplatin.AIM The goal of this study was to examine CIP2A expression and its association with oxaliplatin resistance in human GC cells.METHODS Immunohistochemistry was used to examine CIP2A expression in GC tissues and adjacent normal tissues.CIP2A expression in GC cell lines was reduced using small interfering RNA.After confirming the silencing efficiency,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium and flow cytometry assays were used to evaluate cell proliferation and apoptosis caused by oxaliplatin treatment.Further,the key genes and protein changes were verified using realtime quantitative reverse transcription PCR and Western blotting,respectively,before and after intervention.For bioinformatics analysis,we used the R software and Bioconductor project.For statistical analysis,we used GraphPad Prism 6.0 and the Statistical Package for the Social Sciences software version 20.0(IBM,Armonk,United States).RESULTS A high level of CIP2A expression was associated with tumor size,T stage,lymph node metastasis,Tumor Node Metastasis stage,and a poor prognosis.Further,CIP2A expression was higher in GC cells than in normal human gastric epithelial cells.Using small interfering RNA against CIP2A,we discovered that CIP2A knockdown inhibited cell proliferation and significantly increased GC cell sensitivity to oxaliplatin.Moreover,CIP2A knockdown enhanced oxaliplatin-induced apoptosis in GC cells.Hence,high CIP2A levels in GC may be a factor in chemoresistance to oxaliplatin.In human GC cells,CIP2A regulated protein kinase B phosphorylation,and chemical inhibition of the protein kinase B signaling pathway was significantly associated with increased sensitivity to oxaliplatin.Therefore,the protein kinase B signaling pathway was correlated with CIP2Aenhanced chemoresistance of human GC cells to oxaliplatin.CONCLUSION CIP2A expression could be a novel therapeutic strategy for chemoresistance in GC.展开更多
The serine/threonine phosphatase (PP1) isoform PP1γ2, predominantly expressed in the testis, is a key enzyme in spermatozoa. High PP1γ2 catalytic activity holds motility in check in immature spermatozoa. Inhibitio...The serine/threonine phosphatase (PP1) isoform PP1γ2, predominantly expressed in the testis, is a key enzyme in spermatozoa. High PP1γ2 catalytic activity holds motility in check in immature spermatozoa. Inhibition of PP1γ2 causes motility initiation in immature spermatozoa and motility stimulation and changes in flagellar beat parameters in mature spermatozoa. The PP1γ2 isoform is present in all mammalian spermatozoa studied: mouse, rat, hamster, bovine, non-human primate and man. We have now identified at least four of its regulatory proteins that regulate distinct pools of PP1γ2 within spermatozoa. Our studies provide new insights into biochemical mechanisms underlying development and regulation of sperm motility. We hypothesize that changes in sperm PP1γ2 activity as a result of phosphorylation and reversible binding of the regulatory proteins to the catalytic subunit are critical in the development and regulation of motility and the ability of sperm to fertilize eggs. Targeted disruption of the Ppplcc gene, which encodes the PP1γ1 or PP1γ2 isoforms, causes male infertility in mice as a result of impaired spermiogenesis. Our observations suggest that, in addition to motility, the protein phosphatase PP1γ2 might play an isoform-specific function in the development of specialized flagellar structures of mammalian spermatozoa. (Asian J Androl 2007 July; 9: 445--452)展开更多
Protein phosphatase 2A(PP2A)is a major serine/threonine phosphatase which participates in the regulation of multiple cellular processes.As a confirmed tumor suppressor,PP2A activity is downregulated in tumors and its ...Protein phosphatase 2A(PP2A)is a major serine/threonine phosphatase which participates in the regulation of multiple cellular processes.As a confirmed tumor suppressor,PP2A activity is downregulated in tumors and its re-activation can induce apoptosis of cancer cells.In the brains of Alzheimer's disease(AD)patients,decreased PP2A activity also plays a key role in promoting tau hyperphosphorylation and A0 generation.In this review,we discussed compounds aiming at modulating PP2A activity in the treatment of cancer or AD.The upstream factors that inactivate PP2A in diseases have not been fully elucidated and further studies are needed.It will help for the refinement and development of novel and clinically tractable PP2A-targeted compounds or therapies for the treatment of tumor and AD.展开更多
BACKGROUND Protein phosphatase 2 regulatory subunit B''alpha(PPP2R3A)gene has been reported in other tumors,but the influence of PPP2R3A gene expression on the occurrence,development,and prognosis of hepatocel...BACKGROUND Protein phosphatase 2 regulatory subunit B''alpha(PPP2R3A)gene has been reported in other tumors,but the influence of PPP2R3A gene expression on the occurrence,development,and prognosis of hepatocellular carcinoma(HCC)remains unclear.AIM To investigate whether the PPP2R3A gene could be used to predict tumor recurrence and survival of HCC patients after liver transplantation(LT).METHODS Diseased liver tissues of HCC patients after LT were collected as well as their clinical data and follow-up information.The immunohistochemical method was used to detect the expression of PPP2R3A protein in the tissues of 108 patients with primary liver cancer.Theχ2 test was used to analyze the relationship between PPP2R3A protein expression levels and the clinicopathological features of tumors.The Kaplan-Meier method was used to analyze overall postoperative survival.The COX proportional hazard model was used to analyze adverse prognostic factors.RESULTS Immunohistochemistry showed that the PPP2R3A protein was mainly expressed in the cytoplasm of HCC cells.Compared to corresponding peritumoral tissues,expression was higher in HCC tissues(P≤0.001).Correlation analysis showed that high PPP2R3A expression was correlated with preoperative serum alphafetoprotein(AFP)levels(P=0.003),tumor-node-metastasis-t stage(P≤0.001),and envelope invasion(P=0.001).Univariate analysis showed that overall survival(P≤0.001)and recurrence-free survival(P=0.025)of patients with high PPP2R3A expression(≥4 points)were poor compared to those with low expression(<4 points).The overall survival rates or recurrence-free survival rates at 1,2,and 3 years with high PPP2R3A expression were 73%,38%,and 23%or 31%,23%,and 23%,respectively.Multivariate analysis showed that high PPP2R3A expression(hazard ratio=2.900,95%confidence interval:1.411–5.960,P=0.004)was an independent survival risk factor of HCC patients after LT,and it was also an independent predictor of postoperative tumor recurrence.This study also showed in patients with AFP≥400 ng/mL,the overall survival(P≤0.001)and recurrencefree survival(P=0.023)of those with high PPP2R3A expression were significantly worse compared to those with low PPP2R3A expression.When PPP2R3A expression was low,the overall survival rate(P=0.461)or recurrence-free survival rate(P=0.072)after LT in patients with AFP<400 ng/mL and≥400 ng/mL was not significantly difference.The 1,2,and 3 year survival rate of patients with low PPP2R3A expression and AFP<400 ng/mL were 98%,80%,and 69%,respectively,while patients who met Hangzhou criteria had a posttransplant 1,2,and 3 years overall survival rate of 89%,66%,and 55%,respectively.CONCLUSION High expression of PPP2R3A might be a potential marker for predicting poor prognosis of HCC after LT.Combined with serum AFP levels,PPP2R3A might enhance the accuracy of predicting HCC outcome in patients after LT and supplement the efficacy of the Hangzhou criteria.展开更多
This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in sperrnatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple...This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in sperrnatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple tissue samples were analyzed using bioinformatic resources and RT-PCR technique. A specific rabbit polyclonal antibody was prepared by prokaryotic expression of 2810408A11Rik recombinant protein and utilized for animal immunization. Western blotting, immunohistochemistry and immunofluorescence were used to detect the expression and distribution of 2810408A11Rik. The results of the bioinformatic analysi and RT-PCR showed that 2810408A11Rik mRNA was specifically expressed in mouse testis, and 2810408AllRik protein included a protein phosphatase inhibitor domain. Western blotting assays, immunohistochemistry and immunofluorescence confirmed the expression of 2810408A11Rik protein in mouse testis, especially in post-meiosis round and long spermatids, and that it is localized in the acrosome and the post-nucleus area of sperm. Our findings suggest that 2810408A11Rik may play an important role in spermatogenesis, sperm capacitation and fertilization.展开更多
In regenerating liver of mice, marked increase of the activity of phosphotyrosyl protein phosphatase (PTPP) in cytosol was observed. The PTPP activity varied with time and reached the highest level between 24 to 48 ho...In regenerating liver of mice, marked increase of the activity of phosphotyrosyl protein phosphatase (PTPP) in cytosol was observed. The PTPP activity varied with time and reached the highest level between 24 to 48 hours after partial hepatectomy. In H22a cells the PTPP activity found in every subcellular fraction was lower than that of the normal liver. The PTPP activity was mostly concentrated in lysosomes of normal liver, but mainly distributed in nucleus, cytosol and microsome of regenerating liver. In H22a cells PTPP activity seemed distribute evenly. Five similar major PTPP peaks (I-V) were obtained on DEAE cellulose chromatography of cytosols from all three of liver cells studied. However, two additional PTPP peaks, a and b, were also obtained from cytosol of liver.展开更多
Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB le...Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.展开更多
Protein phosphatase 2A(PP2A)is the pre-dominant serine/threonine phosphatase in eukaryotic cells.In the brains of patients with Alzheimer’s disease(AD),decreased PP2A activities were observed,which is suggested to be...Protein phosphatase 2A(PP2A)is the pre-dominant serine/threonine phosphatase in eukaryotic cells.In the brains of patients with Alzheimer’s disease(AD),decreased PP2A activities were observed,which is suggested to be involved in neurofibrillary tangle(NFT)formation,disturbed amyloid precursor protein(APP)secretion and neurodegeneration in AD brain.Based on our research and other previousfindings,decreased PP2Ac level,decreased PP2A holoenzyme composition,increased level of PP2A inhibitors,increased PP2Ac Leu309 demethylation and Tyr307 phosphorylation underlie PP2A inactivation in AD.β-amyloid(Aβ)over-production,estrogen deficiency and impaired homocys-teine metabolism are the possible up-stream factors that inactivate PP2A in AD neurons.Further studies are required to disclose the role of PP2A in Alzheimer’s disease.展开更多
A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice (Oryza sativa L.) varieties. In this study, we generated an F2 population by crossing the large-grain japoni...A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice (Oryza sativa L.) varieties. In this study, we generated an F2 population by crossing the large-grain japonica cultivar CW23 with Peiai 64 (PA64), an elite indica small-grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly-identified in this study. In particular, qGL3-1, a newly-identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine-mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution (C→A) at position 1092 in exon 10, resulting in replacement of Asp (D) in PA64 with Glu (E) in CW23 for the 364th amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near-isogenic line (NIL) for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection.展开更多
Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn2+ has a direct inhibitory effect on PP2A is ...Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn2+ has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn2+ on PP2A and their direct interaction in vitro. The results showed that Zn2+ mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 pmol/L) of Zn2+ inhibited PP2A directly. Further Zn2+-IDA-agarose affinity binding assays showed that Zn2+ bound to and inhibited PP2Ac(51l-270) but not PP2Ac(1.50) or PP2Ac(27.309). Taken together, Zn2+ inhibits PP2A directly through binding to PP2Ac(51-270) in vitro.展开更多
Protein phosphorylation and dephosphorylation are two essential and vital cellular mechanisms that regulate many receptors and enzymes through kinases and phosphatases.Ca^2+- dependent kinases and phosphatases are res...Protein phosphorylation and dephosphorylation are two essential and vital cellular mechanisms that regulate many receptors and enzymes through kinases and phosphatases.Ca^2+- dependent kinases and phosphatases are responsible for controlling neuronal processing;balance is achieved through opposition.During molecular mechanisms of learning and memory,kinases generally modulate positively while phosphatases modulate negatively.This review outlines some of the critical physiological and structural aspects of kinases and phosphatases involved in maintaining postsynaptic structural plasticity.It also explores the link between neuronal disorders and the deregulation of phosphatases and kinases.展开更多
The aim of this study is to investigate the effect of tyrosine kinase Src on Tyrosine 307(Y307)phosphor-ylation,protein phosphatase 2A(PP2A)activity,and on tau phosphorylation.Specific Src siRNA was transfected into c...The aim of this study is to investigate the effect of tyrosine kinase Src on Tyrosine 307(Y307)phosphor-ylation,protein phosphatase 2A(PP2A)activity,and on tau phosphorylation.Specific Src siRNA was transfected into cultured mouse neuroblastoma N2a cells to inhibit the expression of Src protein,and the phosphorylation levels of PP2A Y307 and tau at different sites,as well as PP2A activity were detected at different time points after siRNA transfection.Twelve hours after siRNA transfec-tion,the protein level of Src was dramatically decreased,with decreased PP2A Y307 phosphorylation.However,the total PP2A protein level was also decreased,together with a decreased PP2A activity.Tau was hyperpho-sphorylated at the Ser198/199/202 sites.Multiple factors may be involved in the cellular regulation of PP2A activ-ity.Inhibiting Src expression could induce inactivation of PP2A and tau hyperphosphorylation.展开更多
Regulatory T(T_(reg))cells constitute a dynamic population that is critical in autoimmunity.T_(reg) cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities.However,recent st...Regulatory T(T_(reg))cells constitute a dynamic population that is critical in autoimmunity.T_(reg) cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities.However,recent studies demonstrated that certain inflammatory conditions induce T_(reg) cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells.Therefore,the identification of novel targets crucial to both T_(reg) cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity.In this study,we found that conditional Pp6 knockout(cKO)in T_(reg) cells led to spontaneous autoinflammation,immune cell activation,and diminished levels of FoxP3 in CD4^(+)T cells in mice.Loss of Pp6 in T_(reg) cells exacerbated two classical mouse models of T_(reg)-related autoinflammation.Mechanistically,Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308,leading to impaired FoxP3 expression in T_(reg) cells.In summary,our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling,thus maintaining T_(reg) cell stability and preventing autoimmune diseases.展开更多
NF-kappaB plays a critical role in cell survival,apoptosis,and inflammatory responses.Serine/threoninespecific phosphatases(PPs)represent the second major class of enzymes that catalyze the dephosphorylation of protei...NF-kappaB plays a critical role in cell survival,apoptosis,and inflammatory responses.Serine/threoninespecific phosphatases(PPs)represent the second major class of enzymes that catalyze the dephosphorylation of proteins.The roles of PPs regulating NF-kappaB activities are poorly understood.Here we describe an RNAi-based screen to identify the PPs that involve in regulating NFkappaB signaling.Thirty-four candidate PPs siRNAs were synthesized and primarily screened by NF-kappaB reporter gene assay in HeLa cells.PHLPP,one of the protein phosphatase type 2C family members(PP2C),was identified as a positive regulator of NF-kappaB signaling.Knock-down of PHLPP dramatically attenuated TNFα-stimulated NF-kappaB transcriptional activation.Knockdown of PHLPP led to enhancement of NF-kappaB/p65 nuclear import and retention,but decreased TNFα-induced phosphorylation at Ser276 on p65.This critical phosphorylation was also drastically reduced by knock-down of PKCalpha and Akt1,two important serine/threonine kinases dephosphorylated by PHLPP.The results together suggest that PHLPP-Akt-PKC may represent an important signaling loop that activates NF-kappaB/p65 signaling through critical serine phosphorylation.展开更多
The mitogen-activated protein kinase(MAPK) signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosp...The mitogen-activated protein kinase(MAPK) signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosphatase 1(MKP1) has an inhibitory effect on the p38 MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 sh RNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42(Aβ42). The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha(TNF-α) and interleukin-1β(IL-1β) m RNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase(JNK) expression levels were assessed using western blot assay. Reactive oxygen species(ROS) levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase d UTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.展开更多
This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated ΔSHP-1) and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 wa...This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated ΔSHP-1) and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta(DE3) E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.展开更多
AIMTo evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process.METHODSARP...AIMTo evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process.METHODSARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin (α-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay.RESULTSThe mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. α-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration.CONCLUSIONPTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.展开更多
Oleanolic acid derivatives act as newer protein tyrosine phosphatase 1B (PTP-1B) inhibitors for type 2 diabetes mellitus (T2DM). In order to understand the structural requirement of PTP-1B inhibitors, 52 oleanolic...Oleanolic acid derivatives act as newer protein tyrosine phosphatase 1B (PTP-1B) inhibitors for type 2 diabetes mellitus (T2DM). In order to understand the structural requirement of PTP-1B inhibitors, 52 oleanolic acid derivatives were divided into a training set (34 compounds) and a test set (18 compounds). The highly reliable and predictive 3D-QSAR models were constructed by CoMFA, CoMSIA and topomer CoMFA methods, respectively. The results showed that the cross validated coefficient (q2) and non-cross-validated coefficient (R2) were 0.554 and 0.999 in the CoMFA model, 0.675 and 0.971 in the CoMSIA model, and 0.628 and 0.939 in the topomer CoMFA model, which suggests that three models are robust and have good exterior predictive capabilities. Furthermore, ten novel inhibitors with much higher inhibitory potency were designed. Our design strategy was that (i) the electronegative substituents (Cl, -CH2OH, OH and -CH2Cl) were introduced into the double bond of ring C, (ii) the hydrogen bond acceptor groups (C≡N and N atom), electronegative groups (C≡N, N atom, -COOH and -COOCH3) and bulky substituents (C6H5N) were connected to the C-3 position, which would result in generating potent and selective PTP-1B inhibitors. We expect that the results in this paper have the potential to facilitate the process of design and to develop new potent PTP-1B inhibitors.展开更多
This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(△TC-PTP) and to study immunohistochemically the expression of △TC-PTP in human non-small c...This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(△TC-PTP) and to study immunohistochemically the expression of △TC-PTP in human non-small cell lung cancers. △TC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-△TC-PTP was expressed in E. coli Rosetta ( DE3 ) host cells and puri- fied. The enzymatic characteristics of △TC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant △TC-PTP. Rabbit polyclonal antibody against △TC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against △TC-PTP protein. △TC-PTP gene was correctly cloned, expressed, and purified. The recombinant △TC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of △TC-PTP (76. 92%, 10/13 ) than adenocarcinoma (57.14%, 4/7) and normal lung tissue(20%, 1/5 ). This study represents the first demonstration that △TC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.展开更多
PTPMEGI is an intracellular protein tyrosine phosphatase(PTP), which contains FERM and PDZ domains This study focuses our attention on the expression, purification and characterization of catalytic domain of PTPMEG1...PTPMEGI is an intracellular protein tyrosine phosphatase(PTP), which contains FERM and PDZ domains This study focuses our attention on the expression, purification and characterization of catalytic domain of PTPMEG1 (AMEG1) and preparation of its polyclonal antibody. A cDNA fragment encoding AMEG1 protein(amino acid residues 643-926) was amplified by PCR and then cloned into the pT7-7 vector. Both soluble and insoluble recombinant AMEG1 proteins were observed after induction by IPTG. Soluble AMEG1 was purified via two chromatographic steps, and the purified enzyme was characterized. With para-nitrophenylphosphate(pNPP) as a substrate, AMEG1 exhibited typical enzymatic characteristics of classic PTPs and classical Michaelis-Menten kinetics. Insoluble AMEG1, which was mainly distributed in the inclusion body of E. coli cells extracts, was purified by preparative electrophoresis gel for the preparation of the polyclonal antibody. A rabbit was immunized with AMEG1 purified by preparative electrophoresis to generate anti-AMEG1 antibody. Anti-serum was collected on 28th day after initial injection and purified via affinity chromatography. The purified polyconal antibody displayed a satisfactory titer and sensitivity.展开更多
基金Supported by This work was supported by the Natural Science Foundation of Gansu Province,China,No.17JR5RA272 and No.22JR5RA923the Research Fund Project of The First Hospital of Lanzhou University,No.ldyyyn2021-120,No.ldyyyn2020-98 and No.ldyyyn2021-30.
文摘BACKGROUND Cancerous inhibitor of protein phosphatase 2A(CIP2A)is a newly discovered oncogene.It is an active cell proliferation regulatory factor that inhibits tumor apoptosis in gastric cancer(GC)cells.CIP2A is functionally related to chemoresistance in various types of tumors according to recent studies.The underlying mechanism,however,is unknown.Further,the primary treatment regimen for GC is oxaliplatin-based chemotherapy.Nonetheless,it often fails due to chemoresistance of GC cells to oxaliplatin.AIM The goal of this study was to examine CIP2A expression and its association with oxaliplatin resistance in human GC cells.METHODS Immunohistochemistry was used to examine CIP2A expression in GC tissues and adjacent normal tissues.CIP2A expression in GC cell lines was reduced using small interfering RNA.After confirming the silencing efficiency,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium and flow cytometry assays were used to evaluate cell proliferation and apoptosis caused by oxaliplatin treatment.Further,the key genes and protein changes were verified using realtime quantitative reverse transcription PCR and Western blotting,respectively,before and after intervention.For bioinformatics analysis,we used the R software and Bioconductor project.For statistical analysis,we used GraphPad Prism 6.0 and the Statistical Package for the Social Sciences software version 20.0(IBM,Armonk,United States).RESULTS A high level of CIP2A expression was associated with tumor size,T stage,lymph node metastasis,Tumor Node Metastasis stage,and a poor prognosis.Further,CIP2A expression was higher in GC cells than in normal human gastric epithelial cells.Using small interfering RNA against CIP2A,we discovered that CIP2A knockdown inhibited cell proliferation and significantly increased GC cell sensitivity to oxaliplatin.Moreover,CIP2A knockdown enhanced oxaliplatin-induced apoptosis in GC cells.Hence,high CIP2A levels in GC may be a factor in chemoresistance to oxaliplatin.In human GC cells,CIP2A regulated protein kinase B phosphorylation,and chemical inhibition of the protein kinase B signaling pathway was significantly associated with increased sensitivity to oxaliplatin.Therefore,the protein kinase B signaling pathway was correlated with CIP2Aenhanced chemoresistance of human GC cells to oxaliplatin.CONCLUSION CIP2A expression could be a novel therapeutic strategy for chemoresistance in GC.
文摘The serine/threonine phosphatase (PP1) isoform PP1γ2, predominantly expressed in the testis, is a key enzyme in spermatozoa. High PP1γ2 catalytic activity holds motility in check in immature spermatozoa. Inhibition of PP1γ2 causes motility initiation in immature spermatozoa and motility stimulation and changes in flagellar beat parameters in mature spermatozoa. The PP1γ2 isoform is present in all mammalian spermatozoa studied: mouse, rat, hamster, bovine, non-human primate and man. We have now identified at least four of its regulatory proteins that regulate distinct pools of PP1γ2 within spermatozoa. Our studies provide new insights into biochemical mechanisms underlying development and regulation of sperm motility. We hypothesize that changes in sperm PP1γ2 activity as a result of phosphorylation and reversible binding of the regulatory proteins to the catalytic subunit are critical in the development and regulation of motility and the ability of sperm to fertilize eggs. Targeted disruption of the Ppplcc gene, which encodes the PP1γ1 or PP1γ2 isoforms, causes male infertility in mice as a result of impaired spermiogenesis. Our observations suggest that, in addition to motility, the protein phosphatase PP1γ2 might play an isoform-specific function in the development of specialized flagellar structures of mammalian spermatozoa. (Asian J Androl 2007 July; 9: 445--452)
基金grants from National Natural Science Foundation of China(No.31970964 and No.31771189)Natural Science Foundation of Hubei Province,China(No.2017CFA065)Wuhan Health and Family Planning Commission(No.WX18C17).
文摘Protein phosphatase 2A(PP2A)is a major serine/threonine phosphatase which participates in the regulation of multiple cellular processes.As a confirmed tumor suppressor,PP2A activity is downregulated in tumors and its re-activation can induce apoptosis of cancer cells.In the brains of Alzheimer's disease(AD)patients,decreased PP2A activity also plays a key role in promoting tau hyperphosphorylation and A0 generation.In this review,we discussed compounds aiming at modulating PP2A activity in the treatment of cancer or AD.The upstream factors that inactivate PP2A in diseases have not been fully elucidated and further studies are needed.It will help for the refinement and development of novel and clinically tractable PP2A-targeted compounds or therapies for the treatment of tumor and AD.
基金National Natural Science Foundation of China,No.81372595.
文摘BACKGROUND Protein phosphatase 2 regulatory subunit B''alpha(PPP2R3A)gene has been reported in other tumors,but the influence of PPP2R3A gene expression on the occurrence,development,and prognosis of hepatocellular carcinoma(HCC)remains unclear.AIM To investigate whether the PPP2R3A gene could be used to predict tumor recurrence and survival of HCC patients after liver transplantation(LT).METHODS Diseased liver tissues of HCC patients after LT were collected as well as their clinical data and follow-up information.The immunohistochemical method was used to detect the expression of PPP2R3A protein in the tissues of 108 patients with primary liver cancer.Theχ2 test was used to analyze the relationship between PPP2R3A protein expression levels and the clinicopathological features of tumors.The Kaplan-Meier method was used to analyze overall postoperative survival.The COX proportional hazard model was used to analyze adverse prognostic factors.RESULTS Immunohistochemistry showed that the PPP2R3A protein was mainly expressed in the cytoplasm of HCC cells.Compared to corresponding peritumoral tissues,expression was higher in HCC tissues(P≤0.001).Correlation analysis showed that high PPP2R3A expression was correlated with preoperative serum alphafetoprotein(AFP)levels(P=0.003),tumor-node-metastasis-t stage(P≤0.001),and envelope invasion(P=0.001).Univariate analysis showed that overall survival(P≤0.001)and recurrence-free survival(P=0.025)of patients with high PPP2R3A expression(≥4 points)were poor compared to those with low expression(<4 points).The overall survival rates or recurrence-free survival rates at 1,2,and 3 years with high PPP2R3A expression were 73%,38%,and 23%or 31%,23%,and 23%,respectively.Multivariate analysis showed that high PPP2R3A expression(hazard ratio=2.900,95%confidence interval:1.411–5.960,P=0.004)was an independent survival risk factor of HCC patients after LT,and it was also an independent predictor of postoperative tumor recurrence.This study also showed in patients with AFP≥400 ng/mL,the overall survival(P≤0.001)and recurrencefree survival(P=0.023)of those with high PPP2R3A expression were significantly worse compared to those with low PPP2R3A expression.When PPP2R3A expression was low,the overall survival rate(P=0.461)or recurrence-free survival rate(P=0.072)after LT in patients with AFP<400 ng/mL and≥400 ng/mL was not significantly difference.The 1,2,and 3 year survival rate of patients with low PPP2R3A expression and AFP<400 ng/mL were 98%,80%,and 69%,respectively,while patients who met Hangzhou criteria had a posttransplant 1,2,and 3 years overall survival rate of 89%,66%,and 55%,respectively.CONCLUSION High expression of PPP2R3A might be a potential marker for predicting poor prognosis of HCC after LT.Combined with serum AFP levels,PPP2R3A might enhance the accuracy of predicting HCC outcome in patients after LT and supplement the efficacy of the Hangzhou criteria.
基金supported by the National 973 Project of China (No.2011CB944304)
文摘This study investigated the expression and distribution of 2810408A11Rik in mouse testis and sperm, and explored its role in sperrnatogenesis and sperm function. The expression levels of 2810408A11Rik mRNA in multiple tissue samples were analyzed using bioinformatic resources and RT-PCR technique. A specific rabbit polyclonal antibody was prepared by prokaryotic expression of 2810408A11Rik recombinant protein and utilized for animal immunization. Western blotting, immunohistochemistry and immunofluorescence were used to detect the expression and distribution of 2810408A11Rik. The results of the bioinformatic analysi and RT-PCR showed that 2810408A11Rik mRNA was specifically expressed in mouse testis, and 2810408AllRik protein included a protein phosphatase inhibitor domain. Western blotting assays, immunohistochemistry and immunofluorescence confirmed the expression of 2810408A11Rik protein in mouse testis, especially in post-meiosis round and long spermatids, and that it is localized in the acrosome and the post-nucleus area of sperm. Our findings suggest that 2810408A11Rik may play an important role in spermatogenesis, sperm capacitation and fertilization.
文摘In regenerating liver of mice, marked increase of the activity of phosphotyrosyl protein phosphatase (PTPP) in cytosol was observed. The PTPP activity varied with time and reached the highest level between 24 to 48 hours after partial hepatectomy. In H22a cells the PTPP activity found in every subcellular fraction was lower than that of the normal liver. The PTPP activity was mostly concentrated in lysosomes of normal liver, but mainly distributed in nucleus, cytosol and microsome of regenerating liver. In H22a cells PTPP activity seemed distribute evenly. Five similar major PTPP peaks (I-V) were obtained on DEAE cellulose chromatography of cytosols from all three of liver cells studied. However, two additional PTPP peaks, a and b, were also obtained from cytosol of liver.
基金supported by Research Start-up Funding of Shenzhen Traditional Chinese Medicine Hospital,No.2021-07(to FB)Sanming Project of Medicine in Shenzhen,No.SZZYSM 202111011(to XDQ and FB)+1 种基金Key Discipline Established by Zhejiang Province,Jiaxing City Jointly-Pain Medicine,No.2019-ss-ttyx(to LSX)Jiaxing Key Laboratory of Neurology and Pain Medicine,No.[2014]81(to LSX)。
文摘Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.
基金supported partially by the National Natural Science Foundation of China(Grant Nos.30500188,30871035 and 30731160621).
文摘Protein phosphatase 2A(PP2A)is the pre-dominant serine/threonine phosphatase in eukaryotic cells.In the brains of patients with Alzheimer’s disease(AD),decreased PP2A activities were observed,which is suggested to be involved in neurofibrillary tangle(NFT)formation,disturbed amyloid precursor protein(APP)secretion and neurodegeneration in AD brain.Based on our research and other previousfindings,decreased PP2Ac level,decreased PP2A holoenzyme composition,increased level of PP2A inhibitors,increased PP2Ac Leu309 demethylation and Tyr307 phosphorylation underlie PP2A inactivation in AD.β-amyloid(Aβ)over-production,estrogen deficiency and impaired homocys-teine metabolism are the possible up-stream factors that inactivate PP2A in AD neurons.Further studies are required to disclose the role of PP2A in Alzheimer’s disease.
基金supported by grants from the Geneti-cally Modified Organisms Breeding Major Projects of China(2011ZX08001-004-009)the National Natural Science Foun-dation of China(30900881)+2 种基金the Jiangxi Province Major Science and Technology Projects and Super Hybrid Rice BreedingDemonstration and Dissemination Projects(20114ABF03105)the Natural Science Foundation of Shandong Province(Y2006D22)
文摘A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice (Oryza sativa L.) varieties. In this study, we generated an F2 population by crossing the large-grain japonica cultivar CW23 with Peiai 64 (PA64), an elite indica small-grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly-identified in this study. In particular, qGL3-1, a newly-identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine-mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution (C→A) at position 1092 in exon 10, resulting in replacement of Asp (D) in PA64 with Glu (E) in CW23 for the 364th amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near-isogenic line (NIL) for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection.
基金supported by Natural Science Foundation of Hubei Province, China (2012FFA044, 2013CFB258)the National Natural Science Foundation of China (81271403, 81471304, 81261120570)
文摘Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn2+ has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn2+ on PP2A and their direct interaction in vitro. The results showed that Zn2+ mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 pmol/L) of Zn2+ inhibited PP2A directly. Further Zn2+-IDA-agarose affinity binding assays showed that Zn2+ bound to and inhibited PP2Ac(51l-270) but not PP2Ac(1.50) or PP2Ac(27.309). Taken together, Zn2+ inhibits PP2A directly through binding to PP2Ac(51-270) in vitro.
文摘Protein phosphorylation and dephosphorylation are two essential and vital cellular mechanisms that regulate many receptors and enzymes through kinases and phosphatases.Ca^2+- dependent kinases and phosphatases are responsible for controlling neuronal processing;balance is achieved through opposition.During molecular mechanisms of learning and memory,kinases generally modulate positively while phosphatases modulate negatively.This review outlines some of the critical physiological and structural aspects of kinases and phosphatases involved in maintaining postsynaptic structural plasticity.It also explores the link between neuronal disorders and the deregulation of phosphatases and kinases.
基金supported by the National Natural Science Foundation of China(Grant Nos.30500188,30471922 and 30731160621)Gamla Tja¨narinnor Foundation and Gun och Bertil Stohnes Stiftelse.
文摘The aim of this study is to investigate the effect of tyrosine kinase Src on Tyrosine 307(Y307)phosphor-ylation,protein phosphatase 2A(PP2A)activity,and on tau phosphorylation.Specific Src siRNA was transfected into cultured mouse neuroblastoma N2a cells to inhibit the expression of Src protein,and the phosphorylation levels of PP2A Y307 and tau at different sites,as well as PP2A activity were detected at different time points after siRNA transfection.Twelve hours after siRNA transfec-tion,the protein level of Src was dramatically decreased,with decreased PP2A Y307 phosphorylation.However,the total PP2A protein level was also decreased,together with a decreased PP2A activity.Tau was hyperpho-sphorylated at the Ser198/199/202 sites.Multiple factors may be involved in the cellular regulation of PP2A activ-ity.Inhibiting Src expression could induce inactivation of PP2A and tau hyperphosphorylation.
基金This work is supported by grants from the National Natural Science Foundation of China(No.82070509,81930088,81725018)Innovative Research Team of High-Level Local Universities in Shanghai,and the Natural Science Foundation of Shanghai Science and Technology Committee,China(No.20ZR1447400).
文摘Regulatory T(T_(reg))cells constitute a dynamic population that is critical in autoimmunity.T_(reg) cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities.However,recent studies demonstrated that certain inflammatory conditions induce T_(reg) cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells.Therefore,the identification of novel targets crucial to both T_(reg) cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity.In this study,we found that conditional Pp6 knockout(cKO)in T_(reg) cells led to spontaneous autoinflammation,immune cell activation,and diminished levels of FoxP3 in CD4^(+)T cells in mice.Loss of Pp6 in T_(reg) cells exacerbated two classical mouse models of T_(reg)-related autoinflammation.Mechanistically,Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308,leading to impaired FoxP3 expression in T_(reg) cells.In summary,our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling,thus maintaining T_(reg) cell stability and preventing autoimmune diseases.
基金This research was supported by the National High Technology Research and Development Program of China(863 Program)(No.2006AA02Z191),the Bureau of Science and Technology of Guangzhou,China(No.2007Z1-E4041)Guangzhou Economic&Technological Development District(GETDD S&T Project)(2007G-P029).
文摘NF-kappaB plays a critical role in cell survival,apoptosis,and inflammatory responses.Serine/threoninespecific phosphatases(PPs)represent the second major class of enzymes that catalyze the dephosphorylation of proteins.The roles of PPs regulating NF-kappaB activities are poorly understood.Here we describe an RNAi-based screen to identify the PPs that involve in regulating NFkappaB signaling.Thirty-four candidate PPs siRNAs were synthesized and primarily screened by NF-kappaB reporter gene assay in HeLa cells.PHLPP,one of the protein phosphatase type 2C family members(PP2C),was identified as a positive regulator of NF-kappaB signaling.Knock-down of PHLPP dramatically attenuated TNFα-stimulated NF-kappaB transcriptional activation.Knockdown of PHLPP led to enhancement of NF-kappaB/p65 nuclear import and retention,but decreased TNFα-induced phosphorylation at Ser276 on p65.This critical phosphorylation was also drastically reduced by knock-down of PKCalpha and Akt1,two important serine/threonine kinases dephosphorylated by PHLPP.The results together suggest that PHLPP-Akt-PKC may represent an important signaling loop that activates NF-kappaB/p65 signaling through critical serine phosphorylation.
文摘The mitogen-activated protein kinase(MAPK) signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosphatase 1(MKP1) has an inhibitory effect on the p38 MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 sh RNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42(Aβ42). The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha(TNF-α) and interleukin-1β(IL-1β) m RNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase(JNK) expression levels were assessed using western blot assay. Reactive oxygen species(ROS) levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase d UTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.
基金the Fund of Science & Technology Bureau of Jilin Province, China(Nos.20060563, 200705394 and 20080434).
文摘This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated ΔSHP-1) and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta(DE3) E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.
基金Supported by Shandong Provincial Natural Science Foundation,China(No.ZR2012HQ004)the Research Fund for Fundamental Research Project of Qingdao(No.13-1-4-180-jch)+1 种基金the Scientific Research Fund of Huangdao District of Qingdao City(No.2014-1-74)the Young People Scientific Research Fund of Affiliated Hospital,Qingdao University(No.QDFY134)
文摘AIMTo evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process.METHODSARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin (α-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay.RESULTSThe mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. α-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration.CONCLUSIONPTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.
基金Supported by the Natural Science Foundation of Guangxi Province(Nos.2013GXNSFAA019019 and 2013GXNSFAA019041)
文摘Oleanolic acid derivatives act as newer protein tyrosine phosphatase 1B (PTP-1B) inhibitors for type 2 diabetes mellitus (T2DM). In order to understand the structural requirement of PTP-1B inhibitors, 52 oleanolic acid derivatives were divided into a training set (34 compounds) and a test set (18 compounds). The highly reliable and predictive 3D-QSAR models were constructed by CoMFA, CoMSIA and topomer CoMFA methods, respectively. The results showed that the cross validated coefficient (q2) and non-cross-validated coefficient (R2) were 0.554 and 0.999 in the CoMFA model, 0.675 and 0.971 in the CoMSIA model, and 0.628 and 0.939 in the topomer CoMFA model, which suggests that three models are robust and have good exterior predictive capabilities. Furthermore, ten novel inhibitors with much higher inhibitory potency were designed. Our design strategy was that (i) the electronegative substituents (Cl, -CH2OH, OH and -CH2Cl) were introduced into the double bond of ring C, (ii) the hydrogen bond acceptor groups (C≡N and N atom), electronegative groups (C≡N, N atom, -COOH and -COOCH3) and bulky substituents (C6H5N) were connected to the C-3 position, which would result in generating potent and selective PTP-1B inhibitors. We expect that the results in this paper have the potential to facilitate the process of design and to develop new potent PTP-1B inhibitors.
基金Supported by the Fund of Science and Technology Department of Jilin Province(No20060563)in part by the Fund of Scienceand Technology Department of Jilin Province(No200505120) the Bureau of Science and Technology of Changchun City(No06GG110)
文摘This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(△TC-PTP) and to study immunohistochemically the expression of △TC-PTP in human non-small cell lung cancers. △TC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-△TC-PTP was expressed in E. coli Rosetta ( DE3 ) host cells and puri- fied. The enzymatic characteristics of △TC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant △TC-PTP. Rabbit polyclonal antibody against △TC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against △TC-PTP protein. △TC-PTP gene was correctly cloned, expressed, and purified. The recombinant △TC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of △TC-PTP (76. 92%, 10/13 ) than adenocarcinoma (57.14%, 4/7) and normal lung tissue(20%, 1/5 ). This study represents the first demonstration that △TC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.
基金Supported by the Plan of Development for Science & Technology of Jilin Province,China(No.20090920)
文摘PTPMEGI is an intracellular protein tyrosine phosphatase(PTP), which contains FERM and PDZ domains This study focuses our attention on the expression, purification and characterization of catalytic domain of PTPMEG1 (AMEG1) and preparation of its polyclonal antibody. A cDNA fragment encoding AMEG1 protein(amino acid residues 643-926) was amplified by PCR and then cloned into the pT7-7 vector. Both soluble and insoluble recombinant AMEG1 proteins were observed after induction by IPTG. Soluble AMEG1 was purified via two chromatographic steps, and the purified enzyme was characterized. With para-nitrophenylphosphate(pNPP) as a substrate, AMEG1 exhibited typical enzymatic characteristics of classic PTPs and classical Michaelis-Menten kinetics. Insoluble AMEG1, which was mainly distributed in the inclusion body of E. coli cells extracts, was purified by preparative electrophoresis gel for the preparation of the polyclonal antibody. A rabbit was immunized with AMEG1 purified by preparative electrophoresis to generate anti-AMEG1 antibody. Anti-serum was collected on 28th day after initial injection and purified via affinity chromatography. The purified polyconal antibody displayed a satisfactory titer and sensitivity.