phosphorylation

摘要： Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex ProTM Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.

摘要： Neurite degeneration,a major component of many neurodegenerative diseases,such as Parkinson’s disease,Alzheimer’s disease,and amyotrophic lateral sclerosis,is not part of the typical apoptosis signaling mechanism,but rather it appears that a self-destructive process is in action.Oxidative stress is a well-known inducer of neurodegenerative pathways:neuronal cell death and neurite degeneration.Although oxidative stress exerts cytotoxic effects leading to neuronal loss,the pathogenic mechanisms and precise signaling pathways by which oxidative stress causes neurite degeneration have remained entirely unknown.We previously reported that reactive oxygen species generated by NADPH oxidases induce activation of the E3 ubiquitin ligase ZNRF1 in neurons,which promotes neurite degeneration.In this process,the phosphorylation of an NADPH oxidase subunit p47-phox at the 345serine residue serves as an important checkpoint to initiate the ZNRF1-dependent neurite degeneration.Evidence provides new insights into the mechanism of reactive oxygen species-mediated neurodegeneration.In this review,we focus specifically on reactive oxygen species-induced neurite degeneration by highlighting a phosphorylation-dependent regulation of the molecular interaction between ZNRF1 and the NADPH oxidase complex.

摘要： Nitrate reductase(NR) is a key enzyme for nitrogen assimilation in plants,and its activity is regulated by posttranslational phosphorylation.To investigate the effects of dephosphorylation of the NIA1 protein on the growth and the physiological and biochemical characteristics of rice under different forms of nitrogen supplies,the phenotypes,nitrogen metabolism and reactive oxygen metabolism were measured in NIA1 phosphorylation site-directed mutant lines(S532 D and S532 A),an Os Nia1 over-expression line(OE) and Kitaake(wild type,WT).Compared with WT and OE,S532 D and S532 A have stronger nitrogen assimilation capacities.When ammonium nitrate served as the nitrogen source,the plant heights,dry weights of shoots and chlorophyll(Chl) contents of S532 D and S532 A were lower than those of the WT and OE,whereas hydrogen peroxide(H_(2)O_(2)),malondialdehyde(MDA) and nitrite contents were higher.When potassium nitrate served as the nitrogen source,the plant heights,dry weights of shoots and Chl contents of S532 D and S532 A were higher than those of the WT and OE,there were no significant differences in the contents of H_(2)O_(2) and MDA in the leaves of the test materials,and the difference in nitrite contents among different lines decreased.When ammonium sulfate served as the nitrogen source,there were no significant differences in the physiological indexes of the test materials,except NR activity.Compared with ammonium nitrate and ammonium sulfate,the content of NH_(4)^(+)-N in the leaves of each plant was lower when potassium nitrate was used as the nitrogen source.The q PCR results showed that Os GS and Os NGS1 were negatively regulated by downstream metabolites,and Os Nrt2.2 was induced by nitrate.In summary,when ammonium nitrate served as the nitrogen source,the weak growth of NIA1 phosphorylation site-directed mutant lines was due to the toxicity caused by the excessive accumulation of nitrite.When potassium nitrate served as the nitrogen source,the assimilation rates of nitrate,nitrite and ammonium salt were accelerated in NIA1 phosphorylation site-directed mutant lines,which could provide more nitrogen nutrition and improve the tolerance of rice to ammonium nitrogen deficiency.These results could provide a possible method to improve the efficiency of nitrogen utilization in rice under low-nitrogen conditions.

摘要： The pre-mRNA processing factor Prp6 is an essential component of the U4/U6.U5 tri-small nuclear ribonucleoprotein(snRNP).In a previous study,mutations were identified in the PRP6 ortholog in four suppressors of Fgprp4 that was deleted of the only kinase FgPrp4 among the spliceosome components in the plant pathogenic fungus Fusarium graminearum.In this study,we identified additional suppressor mutations in FgPrp6 and determined the suppressive effects of selected mutations.In total,12 mutations of FgPRP6 were identified in 20 suppressors of Fgprp4 by sequencing analysis.Whereas three mutation sites are in the linker region of FgPrp6,seven are in the first two HAT repeats.RNA-seq analysis showed that suppressor mutations on different sites caused different splicing efficiency recovery.The suppressive effects of E308K and R230H were verified.Similar to human and fission yeast,the FgPrp6 was phosphorylated by the FgPrp4 kinase.Interestingly,the conserved Prp4-phosphorylation sites T261,T219&T221,and predicted phosphorylation sites T199&T200 on FgPrp6 were dispensable for the function of FgPrp6 in hyphal growth and sexual reproduction but important in plant infection.They are required for the infectious growth of F.graminearum in wheat lemma.RNA-seq analysis of the wheat lemma infected with Fgprp6/FgPRP6^(Δ199-221)-GFP or Fgprp6/FgPRP6^(Δ250-262)-GFP showed that 28 and 35% introns had splicing defects,respectively,which may be responsible for their defects in plant infection.

摘要： Cell extrusion is an active mechanism to eliminate non-viable or supernumerary cells in healthy epithelia.It also plays a role in carcinogenesis,both in tumor growth(apical extrusion)and metastasis(basal extrusion).Embryonic tissues like the neuroepithelium,on the other hand,present rates of proliferation comparable to that of carcinomas,without the occurrence of cell extrusion.However,the downregulation or phosphorylation of actinmodulating proteins like MARCKS,causes extensive neuroepithelial apical cell extrusion.As changes in MARCKS proteins phosphorylation and expression have also been correlated to carcinogenesis,we propose here an integrated model for their functions in epithelial integrity.

摘要： The mitotic exit network(MEN)pathway is a vital kinase cascade regulating the timely and correct progress of cell division.In the rice blast fungus Magnaporthe oryzae,the MEN pathway,consisting of conserved protein kinases MoSep1 and MoMob1-MoDbf2,is important in the development and pathogenicity of the fungus.We found that deletion of MoSEP1 affects the phosphorylation of MoMob1,but not MoDbf2,in contrast to what was found in the buddy yeast Saccharomyces cerevisiae,and verified this finding by in vitro phosphorylation assay and mass spectrometry(MS)analysis.We also found that S43 residue is the critical phosphor-site of MoMob1 by MoSep1,and proved that MoSep1-dependent MoMob1 phosphorylation is essential for cell division during the development of M.oryzae.We further provided evidence demonstrating that MoSep1 phosphorylates MoMob1 to maintain the cell cycle during vegetative growth and infection.Taken together,our results revealed that the MEN pathway has both distinct and conservative functions in regulating the cell cycle during the development and pathogenesis of M.oryzae.

摘要： Mutations in viral proteins can lead to the cold adaption of influenza A virus and the cold-adapted virus is an important vaccination instrument.Here,we identify a novel strain of influenza A virus with cold sensitivity conferred by a mutation at a phosphorylation site within the nucleoprotein(NP).The highly conserved tyrosine 385 residue(Y385)of NP was identified as a phosphorylation site by mass spectrometry.The constructive NP phosphorylation mimicked by Y385 E mutation was fatal for virus replication,while the continuous Y385 dephosphorylation mimicked by Y385 F mutation had little impact on virus replication in vitro.Notably,the Y385 F virus showed much lower replicative capacity in turbinates of mice compared with the wild type virus.Moreover,the replication of Y385 F virus was significantly reduced in both A549 and MDCK cells grown at 33°C,when compared to that at 37°C.These results indicated that the Y385 F mutation led to cold sensitivity of virus.We further found that the cold sensitivity of Y385 F virus could be attributed to diminished NP oligomerization rather than any changes in intracellular localization.Taken together,these findings suggest that the phosphorylation of NP may be a critical factor that regulates the temperature sensitivity of influenza A virus.

摘要： Point mutations in SPG4,the gene encoding spastin,are a frequent cause of hereditary spastic paraplegia(HSP).In some complex HSP cases,there is cognitive impairment.Dynamic trafficking of AMPA receptors into and out of synapses is a key determinant of synaptic plasticity and further implicates learning and memory.However,the exact molecular mechanisms underlying AMPARs trafficking still remains unclear.Here,we performed immunofluorescence staining,whole-cell patch clamp,GST pull-down,and CoIP assays.First,we found that the desumoylation mutant at 427-site of spastin lost its microtubule severing function.Second,our results revealed the maturation of dendritic spines and the increase of GluA2 membrane trafficking after overexpression de-SUMO spastin in cultured hippocampal neurons.Also,we found that the interaction between de SUMO spastin and the GluA2 subunits increased in vitro and in vivo,which were enhanced by the 210-site phosphorylation of spastin at the mean time.Similarly,the phosphorylation modifications enhanced the maturation of dendritic spines and the increasing membrane GluA2 caused by de SUMO spastin.Thus,we show the reciprocal regulation between the phosphorylation and sumoylation of spastin,and jointly modulate the AMPA receptor GluA2 subunits trafficking in cultured hippocampal neurons.

摘要： Phosphoaspartate is one of the major components of eukaryotes and prokaryotic two-component signaling pathways,and it communicates the signal from the sensor of histidine kinase,through the response regulator,to the DNA alongside transcription features and initiates the transcription of correct response genes.Thus,the prediction of phosphoaspartate sites is critical,and its experimental identification can be expensive,time-consuming,and tedious.For this purpose,we propose iPhosD-PseAAC,a new computational model for predicting phosphoaspartate sites in a particular protein sequence using Chou’s 5-steps rues:(1)Benchmark dataset.(2)The feature extraction techniques such as pseudo amino acid composition(PseAAC),statistical moments,and position relative features.(3)For the classification,artificial neural network AAN will be used.(4)In this step,10-fold cross-validation and self-consistency testing will be used for validation.For self-consistency testing,100%Acc is achieved,whereas,for 10-fold crossvalidation 95.14%Acc,95.58%Sn,94.70%Sp and 0.95 MCC are observed.(5).The final step is the development of a user-friendly web server for the ease of users.Thus,the iPhosD-PseAAC is the first and novel predictor for accurate and efficient identification of phosphoaspartate sites.