| In vivo,Secretion via vesicle exocytosis is a fundamental biological event involved in almost all physiological processes,such as synaptic transmission,immune response and glucose homeostasis.The research on the mechanism of cellular vesicle transport and regulation won the Nobel Prize in Physiology in 2013,but the role of phosphorylation regulation in vesicle secretion is not fully understood.Phosphorylation mainly consists of two types,serine/threonine phosphorylation and tyrosine phosphorylation,which are co-regulated by kinases and phosphatases.The involvement of serine/threonine phosphorylation in vesicle secretion during exocytosis has been partially demonstrated,but the precise role of tyrosine phosphorylation in vesicle secretion and which tyrosine phosphatases are involved in the regulation of vesicle secretion are still poorly understood.Here,we proved that megakaryocyte protein tyrosine phosphatase-2(PTP-MEG2)is involved in multiple steps of the process of catecholamine secretion by chromaffin cells.68-kDa PTP-MEG2 encoded by PTPN9 is a typical non-receptor type tyrosine phosphatase.In secretory immune cells,PTP-MEG2 is thought to regulate vesicles fusion process by directly dephosphorizing tyrosine(pY83)at the 83 site of N-ethylbutylimide sensitive fusion protein(NSF).The basic steps and processes of immune cell secretion(or exocytosis)are similar to those of nerve cell secretion.There are many same regulatory proteins in the secretion process,such as snare complex and synaptotagmin.However,they are many differences in the vesicle composition,dynamics of secretion and the molecular mechanism of its regulation.Fewer real-time methods for monitoring immune cell secretion(such as patch clamp recording capacitance,etc.)is explored,the detection is more difficult.Therefore,the study of its mechanism lags behind that of the secretion of nerve cells.Even as nerve cells,there are many differences in the secretory process between the synaptic vesicles and the large dense core vesicles.The role of PTP-MEG2 in the process of vesicle transport,fusion and transmitter release of neuroendocrine cells needs further experimental verification.In addition,although it has been suggested that PTP-MEG2 dephosphorylating NSF causes intracellular vesicle fusion in T cells,the physiological significance of this process is unclear.Because this study did not draw a positive conclusion on whether PTP-MEG2 affects the release of immune factors by T cells.In other words,whether PTP-MEG2 dephosphorylating NSF only affects the intracellular fusion of vesicles(small vesicles become large vesicles),or whether it also affects the release of transmitters in vesicles and other processes in vesicle secretion is still to be studied.Research purposesTo identify the crystal structure of the catalytic domain of PTP-MEG2 and its substrates by structural biological and enzymological studies,to investigate the molecular mechanism of PTP-MEG2 in regulating the secretion of catecholamines in chromaffin cells by electrochemistrical study,to explore the mechanism influence of PTP-MEG2 in regulating the cell vesicle fusion and regulating the kinetics of fusion pores by electrochemistrical and cytological research,to explore whether there are other PTP-MEG2 substrates involved in the secretion regulation processResearch methods1.Plasmid constructionThe sequence of PTP-MEG2 catalytic domain(277-583)was cloned into a pET-15b expression vector with His label at the N-terminal or a pGEX-6P-2 expression vector with GST label at the N-terminal.The full-length of PTP-MEG2 was cloned into a pCDH eukaryotic expression vector with GFP label at the C-terminal.The mutations of PTP-MEG2 were constructed by Stratagene Quikchange kit.2.Protein purificationThe wild-type and mutant catalytic domain proteins of His-tagged PTP-MEG2 were expressed in BL21-DE3 Escherichia coli.Respectively.IPTG was used to induce the protein expression,combine His-tagged PTP-MEG2 by ni-NTA agarose,eluent the binding protein by imidazole,and then gel filtration chromatography was used to purify His-PTP-MEG2.His-PTP-MEG2 catalytic domain proteins of purity over 95%were obtained.GST-MUNC18-1 in BL21-DE3 Escherichia coli.was also expressed under the action of IPTG.After centrifugation and lysis,the proteins were purified by 2 h binding with GST-Sepharose and eluted with GSH3.Determination of enzyme activityThe standard solution(DMG buffer)for our enzymatic reactions is following:50 mM 3,3-dimethyl glutarate pH 7.0,1 mM EDTA,1 mM DTT.The ionic strength was maintained at 0.15 M(adjusted by NaCl).For the pNPP activity measurement,100 ?l reaction mixtures were set up in a total volume in a 96-well polystyrene plate(Thermo Fisher Scientific,Waltham,MA,US).The substrate concentration ranging from 0.2 to 5 Km was used to determine the kcat and Km values.Reactions were started by the addition of an appropriate amount of His-PTP-MEG2-CD-WT or corresponding mutants,such as Y333A,G334R,D335A,Y471A,Y471F,I519A,Q559A,R409A,and R410A.The dephosphorylation of pNPP was terminated by adding 120?l 1M NaOH,and the enzymatic activity was monitored by measuring the absorbance at 405 nm.4.Crystallization and Data CollectionFor crystallization,His-PTP-MEG2-C515A/D470A protein(concentration at 15 mg/ml)was mixed with NSF-pY83 peptide(EVSLpYTFDK)or MUNC18-1-pY145 peptide(ESQVpYSLDS)with molar ratio as 1:3 in buffer A(20 mM HEPES,350 mM NaCl,and 1 mM DTT,pH 7.2).1 ?l mixed protein was blended with 1 ?l buffer B(20%PEG 4000,0.2 M KSCN,10%ethylene glycol,0.1 M bis-tris propane,pH 6.4)at 4?for 3 days before crystals appears.The cubic crystals were preserved in liquid nitrogen very quickly dipped in storage buffer(buffer B supplemented with 10%glycerol).The data were collected at Shanghai Synchrotron Radiation Facility beamline BL17U1 using 0.98A X-ray wavelength and analyzed by HKL2000.5.ElectrochemistryThe catecholamines released by chromaffin cells in the adrenal medulla of mice could be detected by electrochemical method.We used carbon fiber electrodes(5 microns in diameter)to detect the oxidation current peaks formed by the release of catechenamine.Multiclamp 700B amplifier(2012,Axon,Molecular Devices,USA)and version 10.2 of pClamp software were used for electrochemical amperometry.The electrode applies 780 mV voltage and records the ampere current(lamp)at the sampling frequency of 2-5 kHz with the filter of 200 Hz.The electrochemical measurements required the selection of clean and in good condition pheochromochrome cells for electrode proximity.All experiments were recorded at room temperature(20-25?)and we used Igor Software(WareMetrix)for data analysis.6.ImmunofluorescenceIsolate the adrenal medulla of mice(female mice,6-8 weeks).The separated adrenal medulla was immersed in 4%paraformaldehyde and fixed overnight.The fixed tissues were washed in PBS containing 10%sucrose for 4 hours,20%sucrose for 8 hours,and 30%sucrose overnight.The processes of fixation and gradient dehydration were all carried out at 4?.Then these adrenal medullas were imbedded in Tissue-Tek OCT compound and then mounted and frozen them at-25?.Subsequently,the adrenal medulla was cut to 4-?m-thick coronal serial sections.Sections of the adrenal medulla were blocked for 1.5 h.Then,after incubation with primary antibodies and washing with PBS for 3 times,the adrenal medulla were incubated with the secondary antibodies for 1 h at room temperature,stained with DAPI.Images were captured using a confocal microscope(ZEISS,LSM780).The Pearsons co-localization coefficients were analyzed with Image-Pro Plus.7.Quantitative real-time PCRAdrenal medulla was isolated from adult female mice(6-8 weeks).Primary chromaffin cells were infected with lentiviruses encoding wild type or mutant PTP-MEG2,MUNC18-1 or DYNAMIN2.Under fluorescence microscopy,10 to 20 successful infected cells of different brightness were selected from each plate.After the cells were classified according to different brightness,the cells were bonded to the tip of the ultra-fine glass tube,and the glass tube tip with cells was directly smashed in the PCR tube.All the samples were reverse-transcribed using the Revertra Ace qPCR RT Kit(TOYOBO FSQ-101)to obtain cDNA.Then the quantitative real-time PCR was performed with the LightCycler apparatus(Bio-Rad).The expression of actin was used as a control.8.Statistical analysisAll data are presented as mean ± S.E.M.All data were analyzed using two-tailed Student's t-test or one-way ANOVA.All of the Western films were scanned,and band intensity was quantified with ImageJ software(National Institutes of Health,Bethesda MD).P<0.05 was considered as statistically significant.Research results1.PTP-MEG2 regulates multiple steps of exocytosisPTP-MEG2 regulates vesicle fusion and vesicle size during the catecholamine secretion of adrenal gland by modulating the phosphorylation state of the pY83 site of NSF,which relies on the key residues G334,D335(pY loop),Y471(WPD loop),I519(P-loop)and Q559(Q loop).PTP-MEG2 regulates the fusion pore initiation and expansion procedures of catecholamine secretion by the adrenal gland(also designated as foot probability)by modulating the newly identified substrate MUNC18-1 at its pY145 site and DYNAMIN2 at its pY125,through distinct structural basis from that of its regulation of NSF phosphorylation.2.A crystal structure of the PTP-MEG2/phosphor-NSF-pY83 segment was obtainedWe therefore co-crystallized PTP-MEG2/phospho-NSF-E79-pY83-K87,and the structure was solved at 1.7 A resolution.The 2Fo-Fc electron density map allowed for the unambiguous assignment of the phospho-NSF-E79-pY83-K87 in the crystal structure.Importantly,the binding of phospho-NSF-E79-pY83-K87 induced substantial conformational changes in both the WPD loop and ?3-loop-?4 compared to the crystal structure of PTP-MEG2 alone.Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate,a peptide containing phosphorylatthe NSF-pY83 site,specify PTP-MEG2 substrate selectivity and modulate the fusion of catecholamine-containing vesicles.3.Functional delineation of the PTP-MEG2/NSF interface led to the discovery of new PTP-MEG2 substrates.Unexpectedly,delineation of PTP-MEG2 mutants along with the NSF binding interface reveal that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms.Utilizing bioinformatics search and biochemical and electrochemical screening approaches,we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites.Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site.4.PTP-MEG2 regulates fusion pore opening and extension through the DYNAMIN2-pY125 site and MUNC18-1 pY145 site.We found that the tyrosine phosphorylation of MUNC18-1 Y145 site impaired the initial opening of the fusion pore in agonist-induced catecholamine secretion in the primary chromaffin cells.This is probably through either disrupting the arc shape of MUNC18-1 or impairing the interaction between MUNC18-1 and SYNTAXIN1.PTP-MEG2 regulates the opening and expansion of the fusion pore by changing the phosphorylation state of MUNC18-1-pY145.It has been demonstrated that the DYNAMIN2 mediated pore fission process is closely correlated with the receptor endocytosis.In accordance with their effects on percentage of PSF and SAF,overexpression of the DYNAMIN2 wild type or Y125E phospho-mimic mutant markedly increased the Ang?-induced AT1aR endocytosis.The in vitro GTPase activity further suggested that the phosphorylation of DYNAMIN2 at pY125 site up-regulated its activity.Moreover,the LS-MS/MS verified the phosphorylation of DYNAMIN2 at pY125 site in primary adrenal medulla under Ang? stimulation.Collectively,these results indicated that DYNAMIN2 pY125 phosphorylation occurred under Ang? stimulation and negatively regulated the percentage of PSF and SAF of the Ang?-induced catecholamine secretion through increase the GTPase activity of DYNAMIN2.But PTP-MEG2 could reverse this process.5.The distinct structural basis of the recognition of substrates by PTP-MEG2 allows selective inhibitor design.By solving the two crystal structures of PTP-MEG2 in complex with two substrates,the phospho-NSF-E79-pY83-K87 segment and the phospho-MUNC18-1-E141-pY145-S149 segment,we revealed that PTP-MEG2 recognized these functionally different substrates through distinct structural bases.Whereas K411,Y471 and I519 contributed most to the selective interaction of PTP-MEG2 with NSF,another set of residues,including R409 and R410,mediated the specific binding of PTP-MEG2 to MUNC18-1.These data not only indicate that PTP-MEG2 regulates different steps of exocytosis through different substrates in an explicit temporal and spatial context but also afforded important guidance for the design of selective PTP-MEG2 inhibitors according to the different interfaces between PTP-MEG2 and its substrates to explicitly regulate specific physiological processes,supporting the hypothesis of "substrate-specific PTP inhibitors".The design of such inhibitors will certainly help to delineate specific roles of PTP-MEG2 in different physiological and pathological processes.Research conclusionsOur studies demonstrated that PTP-MEG2 regulates two different processes of exocytosis during catecholamine secretion,namely vesicle fusion and the opening and extension of the fusion pore through two distinct structural bases.Whereas the PTP-MEG2 regulated the vesicle fusion via dephosphorylation of NSF by specific contacting residues,PTP-MEG2 regulated pore fusion dynamics through at least two different substrates,the MUNC18-1 and DYNAMIN2,with distinct sets of structural features.The present study also supports the hypothesis that the tyrosine phosphorylation of secretion machinery proteins is an important category of regulatory event for hormone secretion and is explicitly regulated by protein tyrosine phosphatases,such as PTP-MEG2.Research significance1.PTP-MEG2 actively regulates multiple steps of exocytosis and has broader effects on hormone and neurotransmitter secretion,in addition to its previously known role in modulating immunocyte secretion.These broader effects of PTP-MEG2 in exocytosis are consistent with the finding that PTP-MEG2 knockout causes embryonic lethal in mice and obvious defects in neural development were visualized by immunofluorescence deficiency.2.A general substrate recognition motif of PTP-MEG2 was identified,accounting for its substrate specificity.This structural motif includes Y333,G334,D335 and Q559.In particular,the D335:Q559 pair causes PTP-MEG2 to favour a small amino acid at the pY+1 site of its substrate.This information will greatly facilitate the identification of new substrates of PTP-MEG23.Functional delineation of the PTP-MEG2/NSF interface revealed previously unknown PTP-MEG2 substrates.Our studies highlight that the combination of structural determination and functional delineation of the interface mutants of the protein complex is a powerful approach in characterizing signalling events and identifying unknown downstream signalling molecules4.Our study further expands the underlying mechanism of complex exocytosis processes occurring in vivo,and clarifies and enriches the mechanisms of dephosphorylation events in regulating vesicle fusion and the dynamics of fusion pores 5.PTP-MEG2 regulates vesicle fusion and initial opening of the fusion pore via distinct structural bases.These data not only indicate that PTP-MEG2 regulates different steps of exocytosis through different substrates in an explicit temporal and spatial context but also afforded important guidance for the design of selective PTP-MEG2 inhibitors according to the different interfaces between PTP-MEG2 and its substrates to explicitly regulate specific physiological processes,supporting the hypothesis of "substrate-specific PTP inhibitors".The design of such inhibitors will certainly help to delineate specific roles of PTP-MEG2 in different physiological and pathological processes. |
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