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 ...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.展开更多
The limited CO_(2)content in aqueous solution and low adsorption amount of CO_(2)on catalyst surface lead to poor photocatalytic CO_(2)reduction activity and selectivity.Herein,the design and fabrication of a novel ph...The limited CO_(2)content in aqueous solution and low adsorption amount of CO_(2)on catalyst surface lead to poor photocatalytic CO_(2)reduction activity and selectivity.Herein,the design and fabrication of a novel photocatalytic architecture is reported,accomplished via chemical vapor deposition of polymeric carbon nitride on carbon paper.The as-obtained samples with a hydrophobic surface exhibit excellent CO_(2)transport and adsorption ability,as well as the building of triphase air-liquid-solid(CO_(2)-H_(2)O-catalyst)joint interfaces,eventually resulting in the inhibition of H2 evolution and great promotion of CO_(2)reduction with a selectivity of 78.6%.The addition of phosphate to reaction environment makes further improvement of CO_(2)photoreduction into carbon fuels with a selectivity of 93.8%and an apparent quantum yield of 0.4%.This work provides new insight for constructing efficient photocatalytic architecture of CO_(2)photoreduction in aqueous solution and demonstrates that phosphate could play a key role in this process.展开更多
Repair of DNA double-strand break(DSB)is critical for the maintenance of genome integrity.A class of DSB-induced small RNAs(di RNAs)has been shown to play an important role in DSB repair.In humans,di RNAs are associat...Repair of DNA double-strand break(DSB)is critical for the maintenance of genome integrity.A class of DSB-induced small RNAs(di RNAs)has been shown to play an important role in DSB repair.In humans,di RNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination(HR).Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions.However,the role of Ago2 phosphorylation in DNA damage repair is unexplored.Here,we show that S672,S828,T830,and S831 of human Ago2 are phosphorylated in response to ionizing radiation(IR).S672 A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency.We further show that defective association of Ago2 S672 A variant with DSB sites,instead of defects in di RNA and Rad51 binding,may account for decreased Rad51 foci formation and HR efficiency.Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.展开更多
Influenza A virus(IAV)continues to pose a pandemic threat to public health,resulting a high mortality rate annually and during pandemic years.Posttranslational modification of viral protein plays a substantial role in...Influenza A virus(IAV)continues to pose a pandemic threat to public health,resulting a high mortality rate annually and during pandemic years.Posttranslational modification of viral protein plays a substantial role in regulating IAV infection.Here,based on immunoprecipitation(IP)-based mass spectrometry(MS)and purified virus-coupled MS,a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified,including 60 novel phosphorylation sites.Additionally,for the first time,we provide evidence that PB2 can also be acetylated at site K187.Notably,the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS.Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains,suggesting their fundamental importance in the IAV life cycle.Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure.While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice,PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice.Mechanistically,PB2 S181E substantially impairs viral polymerase activity and viral replication,remarkably dampens protein stability and nuclear accumulation of PB2,and significantly weakens IAV-induced inflammatory responses.Therefore,our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins,laying a foundation for subsequent mechanistic studies.Meanwhile,the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.展开更多
Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases...Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases（Src and Fyn）or phosphatases（PTPαand STEP）are involved in regulating the phosphorylation of NMDARs.In this study,we used immunoblotting to investigate the role of an NMDAR subpopulation on the phosphorylation level of the GluN2B subunit at the Y1336 and Y1472sites in rat brain slices after NMDA treatment.We found that NMDA stimulation dramatically decreased the phosphorylation level of GluN2B at Y1472 in a dose-and time-dependent manner,but not at Y1336.Extrasynaptic NMDAR activation did not reduce the phosphorylation of GluN2B at Y1472.In addition,ifenprodil,a selective antagonist of GluN2Bcontaining NMDARs,did not abolish the decreased phosphorylation of GluN2B at Y1472 triggered by NMDA.These results suggest that the activation of synaptic GluN2A-containing NMDARs is required for the decreased phosphorylation of GluN2B at Y1472that is induced by NMDA treatment in rat brain slices.展开更多
Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase, and hence its processing between the endoplasmic reticulum (ER), Golgi and trans-Golgi (TGN) network remains under a strict control of factors that af...Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase, and hence its processing between the endoplasmic reticulum (ER), Golgi and trans-Golgi (TGN) network remains under a strict control of factors that affect the microtubule (MT) stabilization, and the recruitment and activation of coat and cargo proteins, including ADP-ribosylation factors (Arfs) and protein kinase D (PKD). Here, we report on the factors implicated in the regulation of MMP-9 secretion by salivary gland acinar cells in response to P. gingivalis LPS, and the effect of hormone, ghrelin. We show that the LPS-elicited induction in MMP-9 secretion is associated with the increase in α-tubulin acetylation and the enhancement in MT stabilization, while the modulatory effect of ghrelin is reflected in a decrease in α-tubulin acetylation. Further, the effect of the LPS occurs in concert with up-regulation in Arf-guanine nucleotide exchange factor (GEF)-mediated Arf1 activation and the TGN recruitment of PKD2, while ghrelin exerts the modulatory effect on Arf-GEF activation. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. The findings demonstrate that MT stabilization along with Arf-GEF-mediated Arf1/PKD2 activation play a major role in P. gingivalis LPS-induced up-regulation in salivary gland acinar cell MMP-9 secretion, and point the modulatory mode of action by ghrelin.展开更多
BACKGROUND： Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal con...BACKGROUND： Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal conduction and induce a toxic reaction. Organophosphorus insecticides may act on M2 muscarinic acetylcholine receptors, whose combination with G proteins is regulated by phosphorylation of G protein-coupled receptor kinase 2. OBJECTIVE： To investigate the effects of organophosphorus insecticides on the phosphorylation of G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptors and to reveal other possible actions of organophosphorus insecticides. DESIGN, TIME AND SETTING： An observational study, which was performed in the Central Laboratory of Shenyang Medical College, and Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University from June 2002 to December 2004. MATERIALS： Paraoxon, parathion, chlorpyrifos, and chlorpyrifos oxon were provided by Chem Service Company, USA, [γ -p^32] ATP and [^35S]GTP γ S by New England Nuclear Life Science Products, and recombinant β 2-adrenergic receptor membrane protein by Sigma Company, USA. METHODS： The M2 muscarinic acetylcholine receptor was extracted and purified from pig brain using affinity chromatography. Subsequently, the purified M2 muscarinic acetylcholine receptor, G protein-coupled receptor kinase 2, and [γ -p^32] ATP were incubated with different concentrations of paraoxon and chlorpyrifos oxon together. The mixture then underwent polyacrylamide gel electrophoresis, and the gel film was dried and radioactively autographed to detect phosphorylation of the M2 muscarinic acetylcholine receptor. Finally, the radio-labeled phosphorylated M2 receptor protein band was excised for counting with an isotope liquid scintillation counter. MAIN OUTCOME MEASURES： Effects of chlorpyrifos oxon, paraoxon, chlorpyrifos, and parathion in different concentrations on the phosphorylation of the M2 muscarinic acetylcholine receptor; effects of chlorpyrifos oxon on the phosphorylation of the β -adrenergic receptor. RESULTS： Chlorpyrifos oxon could completely inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor, and its IC50 was 70 μ mol/L. Chlorpyrifos could also inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. However, paraoxon and parathion could not inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. Chlorpyrifos oxon in different concentrations could also not inhibit the phosphorylation of the β 2-adrenergic receptor catalyzed by G protein-coupled receptor kinase 2. CONCLUSION： Different kinds of organophosphorus insecticides have different effects on the phosphorylation of the G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptor. Organophosphorus insecticides possibly have different toxic effects.展开更多
In order to further investigate the role of ClC-2（ClC=chloride-ion channel） played in the regulation of cell proliferation and differentiation, the capablity of ClC-2 phosphorylation catalyzed by mitogen-activated p...In order to further investigate the role of ClC-2（ClC=chloride-ion channel） played in the regulation of cell proliferation and differentiation, the capablity of ClC-2 phosphorylation catalyzed by mitogen-activated protein ki-nase（MAPK） was studied. A mutation of 659Ser to Ala（S659A） of the rabbit ClC-2 cDNA in the consensus sequence of MAPK phosphorylation was introduced by overlap extension polymerase chain reaction（PCR）. Recombinant vectors pGEX-4T-1/ClC-2-2CT and pGEX-4T-1/ClC-2CT（S659A） were constructed. They were transformed to E. coli BL21, expressed by isopropy-β-D-thiogalactoside（IPTG） induction, the recombinant proteins were subjected to purification by glutathione sepharose 4B affinity chromatography. In vitro phosphorylation of the fusion proteins catalyzed by MAPK was performed. The results show that fusion protein GST/ClC-2CT（wild type） can be phosphorylated by MAPK, and this phosphorylation can be restrained by the inhibitor p42/44MAPK, PD98095; while the phosphorylation level of fusion protein GST/ClC-2CT（S659A）（mutant） was significantly reduced. Therefore, ClC-2 can be phosphorylated by MAPK and the target site of the phosphorylation is most likely the 659Ser residue.展开更多
The efficient translation of most eukaryotic mRNAs requires an interaction between the 5' m7GTP cap structure of mRNA and eIF-4F which is composed of 25-(eIF 4E),46-(eIF-4A), and 220-kDa (p220) subunits. eIF-4E bi...The efficient translation of most eukaryotic mRNAs requires an interaction between the 5' m7GTP cap structure of mRNA and eIF-4F which is composed of 25-(eIF 4E),46-(eIF-4A), and 220-kDa (p220) subunits. eIF-4E binds specifically to the mRNA cap. Evidence indicates that stimulation of eIF-4E phosphorylation increases the efficiency of the translational initiation by an as yet undefined mechanism. Phosphorylation of both eIF-4E and p220 in intact cells is stimulated by growth factors, and eIF-4E phosphorylation appears to be a critical event during cell growth regulation. To date only serine phosphorylation of eIF-4E has been described in vivo. In these studies we found that treatment of HepG2 cells with okadaic acid resulted in eIF-4E phosphorylation on both serine and threonine residues and that tryptic phosphopeptide maps showed several previously unrecognized phosphopeptides. Analysis of p,,' from control and okadaic acid--treated cells dmonstrated serine and threonine phosphorylation under both conditions. The most notable finding was that hyperphosphorylation of eIF-4E and p220 increased binding of p220 but not eIF-4E to the m7GTP.cap structure. we suggest that phosphorylation of eIF-4E is more complicated than previously recognizes and that hyperphosphorylation of eIF-4E and p220recruits more p220 into the protein complex that associate with mRNA caps.展开更多
Endothelin-1 and a number of other genes expressd primarily in endothelial cells(EC)require a functional GATA element in their promoter region.The widely expressed zinc finger DNA binding protein GATA-2 has been chara...Endothelin-1 and a number of other genes expressd primarily in endothelial cells(EC)require a functional GATA element in their promoter region.The widely expressed zinc finger DNA binding protein GATA-2 has been characterized as the likely GATA factor which binds these GATA elements.To understand the specificity of this interaction,and to investigate the potential for regulation of GATA-2 activity,we have studied translation and post-translational modification of the GATA-2 protein. A specific antiserum immunoprecipitated a 52kDa GATA-2 protein from [35-S] methionine-labeled EC,as well as a wide variety of cultured human cell lines which express GATA-2 mRNA. Immunoprecipitation experiments with [32-P]-orthophosphate labeled cells indicated that GATA-2 is similarly phosphorylated in EC and non-EC lines. Thus the apparent cell-specific activity of this transcription factor is not regulated by translation or phosphorylation, and must derive from the interaction of GATA-2 with other nuclear proteins in the EC.Further studies investigated the potential regulation of GATA-2 phosphorylation in EC. Phosphoamino acid analysis indicated that GATA-2 is phosphorylated on serine and threonine residues in EC.The hasal phosphorylation of GATA-2 was rapidly and markedly increased when EC were treated with calcium ionophore A23187, while phorbol ester and forskolin had no effect.Phosphopeptide map analysis showed that A23187 induced phosphorylation of at least two additional sites in GATA-2.Gel shift assays employing nuclear extracts isolated from EC that had been treated with A23187 had a different DNA binding pattern when compared to control.This regulated phosphorylation of GATA-2 may provide a signaling pathway for hormonal regulation of endothelial cell genes such as endothelin-1 which alter their rate of transcription in response to increased intracellular calcium.展开更多
基金We thank members of our group for insightful discussion during the course of this study.This work was supported by grants from Chinese Academy of Science(KSCX1-YW-R65,KSCX2-YW-H10)National Basic Research Program of China(2002CB713700)+4 种基金Hi-Tech Research and Development Program of China(2001AA215331)Chinese Minister of Education(20020358051 to XY,PCSIRT0413 to XD)National Natural Science Foundation of China(39925018,30270293 to XY,30500183 to XD,30600222 to JY)National Institutes of Health(USA)(DK56292,CA92080)to XY(a Georgia Cancer Coalition Eminent Scholar)JY was supported by China Postdoctor(2005037560).
文摘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.
文摘The limited CO_(2)content in aqueous solution and low adsorption amount of CO_(2)on catalyst surface lead to poor photocatalytic CO_(2)reduction activity and selectivity.Herein,the design and fabrication of a novel photocatalytic architecture is reported,accomplished via chemical vapor deposition of polymeric carbon nitride on carbon paper.The as-obtained samples with a hydrophobic surface exhibit excellent CO_(2)transport and adsorption ability,as well as the building of triphase air-liquid-solid(CO_(2)-H_(2)O-catalyst)joint interfaces,eventually resulting in the inhibition of H2 evolution and great promotion of CO_(2)reduction with a selectivity of 78.6%.The addition of phosphate to reaction environment makes further improvement of CO_(2)photoreduction into carbon fuels with a selectivity of 93.8%and an apparent quantum yield of 0.4%.This work provides new insight for constructing efficient photocatalytic architecture of CO_(2)photoreduction in aqueous solution and demonstrates that phosphate could play a key role in this process.
基金supported by the National Natural Science Foundation of China(31401202)。
文摘Repair of DNA double-strand break(DSB)is critical for the maintenance of genome integrity.A class of DSB-induced small RNAs(di RNAs)has been shown to play an important role in DSB repair.In humans,di RNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination(HR).Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions.However,the role of Ago2 phosphorylation in DNA damage repair is unexplored.Here,we show that S672,S828,T830,and S831 of human Ago2 are phosphorylated in response to ionizing radiation(IR).S672 A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency.We further show that defective association of Ago2 S672 A variant with DSB sites,instead of defects in di RNA and Rad51 binding,may account for decreased Rad51 foci formation and HR efficiency.Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.
基金supported by the National Natural Science Foundation of China(32072832,32372976)by the National Key Research and Development Project of China(2021YFD1800202)+3 种基金by Jiangsu Province Agricultural Science&Technology Independent Innovation Funds[CX(21)3141]by the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX22_3553 and KYCX21_3277)by the Earmarked Fund for China Agriculture Research System(CARS-40)a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘Influenza A virus(IAV)continues to pose a pandemic threat to public health,resulting a high mortality rate annually and during pandemic years.Posttranslational modification of viral protein plays a substantial role in regulating IAV infection.Here,based on immunoprecipitation(IP)-based mass spectrometry(MS)and purified virus-coupled MS,a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified,including 60 novel phosphorylation sites.Additionally,for the first time,we provide evidence that PB2 can also be acetylated at site K187.Notably,the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS.Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains,suggesting their fundamental importance in the IAV life cycle.Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure.While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice,PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice.Mechanistically,PB2 S181E substantially impairs viral polymerase activity and viral replication,remarkably dampens protein stability and nuclear accumulation of PB2,and significantly weakens IAV-induced inflammatory responses.Therefore,our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins,laying a foundation for subsequent mechanistic studies.Meanwhile,the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.
基金supported by grants from the National Natural Science Foundation of China (30900418)the Natural Science Program of Department of Education of Zhejiang Province,China (Y201122469)
文摘Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases（Src and Fyn）or phosphatases（PTPαand STEP）are involved in regulating the phosphorylation of NMDARs.In this study,we used immunoblotting to investigate the role of an NMDAR subpopulation on the phosphorylation level of the GluN2B subunit at the Y1336 and Y1472sites in rat brain slices after NMDA treatment.We found that NMDA stimulation dramatically decreased the phosphorylation level of GluN2B at Y1472 in a dose-and time-dependent manner,but not at Y1336.Extrasynaptic NMDAR activation did not reduce the phosphorylation of GluN2B at Y1472.In addition,ifenprodil,a selective antagonist of GluN2Bcontaining NMDARs,did not abolish the decreased phosphorylation of GluN2B at Y1472 triggered by NMDA.These results suggest that the activation of synaptic GluN2A-containing NMDARs is required for the decreased phosphorylation of GluN2B at Y1472that is induced by NMDA treatment in rat brain slices.
文摘Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase, and hence its processing between the endoplasmic reticulum (ER), Golgi and trans-Golgi (TGN) network remains under a strict control of factors that affect the microtubule (MT) stabilization, and the recruitment and activation of coat and cargo proteins, including ADP-ribosylation factors (Arfs) and protein kinase D (PKD). Here, we report on the factors implicated in the regulation of MMP-9 secretion by salivary gland acinar cells in response to P. gingivalis LPS, and the effect of hormone, ghrelin. We show that the LPS-elicited induction in MMP-9 secretion is associated with the increase in α-tubulin acetylation and the enhancement in MT stabilization, while the modulatory effect of ghrelin is reflected in a decrease in α-tubulin acetylation. Further, the effect of the LPS occurs in concert with up-regulation in Arf-guanine nucleotide exchange factor (GEF)-mediated Arf1 activation and the TGN recruitment of PKD2, while ghrelin exerts the modulatory effect on Arf-GEF activation. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. The findings demonstrate that MT stabilization along with Arf-GEF-mediated Arf1/PKD2 activation play a major role in P. gingivalis LPS-induced up-regulation in salivary gland acinar cell MMP-9 secretion, and point the modulatory mode of action by ghrelin.
文摘BACKGROUND： Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal conduction and induce a toxic reaction. Organophosphorus insecticides may act on M2 muscarinic acetylcholine receptors, whose combination with G proteins is regulated by phosphorylation of G protein-coupled receptor kinase 2. OBJECTIVE： To investigate the effects of organophosphorus insecticides on the phosphorylation of G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptors and to reveal other possible actions of organophosphorus insecticides. DESIGN, TIME AND SETTING： An observational study, which was performed in the Central Laboratory of Shenyang Medical College, and Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University from June 2002 to December 2004. MATERIALS： Paraoxon, parathion, chlorpyrifos, and chlorpyrifos oxon were provided by Chem Service Company, USA, [γ -p^32] ATP and [^35S]GTP γ S by New England Nuclear Life Science Products, and recombinant β 2-adrenergic receptor membrane protein by Sigma Company, USA. METHODS： The M2 muscarinic acetylcholine receptor was extracted and purified from pig brain using affinity chromatography. Subsequently, the purified M2 muscarinic acetylcholine receptor, G protein-coupled receptor kinase 2, and [γ -p^32] ATP were incubated with different concentrations of paraoxon and chlorpyrifos oxon together. The mixture then underwent polyacrylamide gel electrophoresis, and the gel film was dried and radioactively autographed to detect phosphorylation of the M2 muscarinic acetylcholine receptor. Finally, the radio-labeled phosphorylated M2 receptor protein band was excised for counting with an isotope liquid scintillation counter. MAIN OUTCOME MEASURES： Effects of chlorpyrifos oxon, paraoxon, chlorpyrifos, and parathion in different concentrations on the phosphorylation of the M2 muscarinic acetylcholine receptor; effects of chlorpyrifos oxon on the phosphorylation of the β -adrenergic receptor. RESULTS： Chlorpyrifos oxon could completely inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor, and its IC50 was 70 μ mol/L. Chlorpyrifos could also inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. However, paraoxon and parathion could not inhibit the phosphorylation of the M2 muscarinic acetylcholine receptor. Chlorpyrifos oxon in different concentrations could also not inhibit the phosphorylation of the β 2-adrenergic receptor catalyzed by G protein-coupled receptor kinase 2. CONCLUSION： Different kinds of organophosphorus insecticides have different effects on the phosphorylation of the G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptor. Organophosphorus insecticides possibly have different toxic effects.
基金Supported by the National Natural Science Foundation of China(No.30973274)
文摘In order to further investigate the role of ClC-2（ClC=chloride-ion channel） played in the regulation of cell proliferation and differentiation, the capablity of ClC-2 phosphorylation catalyzed by mitogen-activated protein ki-nase（MAPK） was studied. A mutation of 659Ser to Ala（S659A） of the rabbit ClC-2 cDNA in the consensus sequence of MAPK phosphorylation was introduced by overlap extension polymerase chain reaction（PCR）. Recombinant vectors pGEX-4T-1/ClC-2-2CT and pGEX-4T-1/ClC-2CT（S659A） were constructed. They were transformed to E. coli BL21, expressed by isopropy-β-D-thiogalactoside（IPTG） induction, the recombinant proteins were subjected to purification by glutathione sepharose 4B affinity chromatography. In vitro phosphorylation of the fusion proteins catalyzed by MAPK was performed. The results show that fusion protein GST/ClC-2CT（wild type） can be phosphorylated by MAPK, and this phosphorylation can be restrained by the inhibitor p42/44MAPK, PD98095; while the phosphorylation level of fusion protein GST/ClC-2CT（S659A）（mutant） was significantly reduced. Therefore, ClC-2 can be phosphorylated by MAPK and the target site of the phosphorylation is most likely the 659Ser residue.
文摘The efficient translation of most eukaryotic mRNAs requires an interaction between the 5' m7GTP cap structure of mRNA and eIF-4F which is composed of 25-(eIF 4E),46-(eIF-4A), and 220-kDa (p220) subunits. eIF-4E binds specifically to the mRNA cap. Evidence indicates that stimulation of eIF-4E phosphorylation increases the efficiency of the translational initiation by an as yet undefined mechanism. Phosphorylation of both eIF-4E and p220 in intact cells is stimulated by growth factors, and eIF-4E phosphorylation appears to be a critical event during cell growth regulation. To date only serine phosphorylation of eIF-4E has been described in vivo. In these studies we found that treatment of HepG2 cells with okadaic acid resulted in eIF-4E phosphorylation on both serine and threonine residues and that tryptic phosphopeptide maps showed several previously unrecognized phosphopeptides. Analysis of p,,' from control and okadaic acid--treated cells dmonstrated serine and threonine phosphorylation under both conditions. The most notable finding was that hyperphosphorylation of eIF-4E and p220 increased binding of p220 but not eIF-4E to the m7GTP.cap structure. we suggest that phosphorylation of eIF-4E is more complicated than previously recognizes and that hyperphosphorylation of eIF-4E and p220recruits more p220 into the protein complex that associate with mRNA caps.
文摘Endothelin-1 and a number of other genes expressd primarily in endothelial cells(EC)require a functional GATA element in their promoter region.The widely expressed zinc finger DNA binding protein GATA-2 has been characterized as the likely GATA factor which binds these GATA elements.To understand the specificity of this interaction,and to investigate the potential for regulation of GATA-2 activity,we have studied translation and post-translational modification of the GATA-2 protein. A specific antiserum immunoprecipitated a 52kDa GATA-2 protein from [35-S] methionine-labeled EC,as well as a wide variety of cultured human cell lines which express GATA-2 mRNA. Immunoprecipitation experiments with [32-P]-orthophosphate labeled cells indicated that GATA-2 is similarly phosphorylated in EC and non-EC lines. Thus the apparent cell-specific activity of this transcription factor is not regulated by translation or phosphorylation, and must derive from the interaction of GATA-2 with other nuclear proteins in the EC.Further studies investigated the potential regulation of GATA-2 phosphorylation in EC. Phosphoamino acid analysis indicated that GATA-2 is phosphorylated on serine and threonine residues in EC.The hasal phosphorylation of GATA-2 was rapidly and markedly increased when EC were treated with calcium ionophore A23187, while phorbol ester and forskolin had no effect.Phosphopeptide map analysis showed that A23187 induced phosphorylation of at least two additional sites in GATA-2.Gel shift assays employing nuclear extracts isolated from EC that had been treated with A23187 had a different DNA binding pattern when compared to control.This regulated phosphorylation of GATA-2 may provide a signaling pathway for hormonal regulation of endothelial cell genes such as endothelin-1 which alter their rate of transcription in response to increased intracellular calcium.
