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Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP
采用 32P-γATP体外测定VPS34/PIK3C3的活性   

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Abstract

VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al., 2013). Assaying VPS34 activity directly in vitro can be a key stage in delineating the action of a particular stimulus.

Keywords: Phosphatidylinositol-3-phosphate(磷脂酰肌醇3 -磷酸), VPS34(Vps34), PIK3C3(pik3c3), Lipid kinase assay(脂质激酶测定), TLC(薄层色谱法)

Materials and Reagents

  1. Hamilton syringe (500 μl) (Hamilton, catalog number: 80865 )
  2. Pyrex tubes (12 x 75 mm) (Corning, catalog number: 99445-12 )
  3. Eppendorf flex tubes (Eppendorf, catalog number: 022364111 )
  4. Spin-X columns (0.22 μm) (Corning, Costar®, catalog number: 8161 )
  5. Whatman paper (3 mm) (Sigma-Aldrich, catalog number: 3030-917 )
  6. Silica 60 TLC plates (Merck Millipore Corporation, catalog number: 1.05553.0001 )
  7. TLC spotting capillary tubes, 15.2 cm (VWR, catalog number: 80060-608 )
  8. Polycarbonate membranes, 100 nm pore size (Avanti Polar Lipids, catalog number: 610005 )
  9. Chloroform (VWR International, catalog number: 22707.320 )
  10. Crude liver phosphatidylinositol (PI) (Avanti, catalog number: 840042C )
  11. Methanol (VWR International, catalog number: 20847.307 )
  12. N2 gas tank (99.998% Pure) 
  13. VPS34/VPS15 recombinant protein (MRCPPU Reagents, catalog number: DU8692 )
  14. 32P-γ-ATP (PerkinElmer, catalog number: NEG002A500UC )
  15. ATP (Sigma-Aldrich, catalog number: A2383
  16. Hydrochloric acid (37%) (VWR International, catalog number: 20252.335 )
  17. Glycerol (VWR International, catalog number: 24388.320 )
  18. Sodium dodecyl sulphate (SDS) (VWR International, catalog number: 444464T )
  19. Bromophenol blue (Sigma-Aldrich, catalog number: 114405 )
  20. β-mercaptoethanol
  21. Tris base (VWR International, catalog number: 103157P )
  22. Sodium chloride (VWR International, catalog number: 27810.364 )
  23. Manganese chloride (Sigma-Aldrich, catalog number: M3634 )
  24. CHAPS (Sigma-Aldrich, catalog number: C3023 )
  25. Dithiothreitol (DTT) (ForMedium, catalog number: DTT010 )
  26. Ammonium hydroxide (Sigma-Aldrich, catalog number: 320145 )
  27. ATP mix (5x) (see Recipes)
  28. Kinase assay buffer (see Recipes)
  29. Stop solution (see Recipes)
  30. Sample buffer (1x) (see Recipes)
  31. TLC solvent (see Recipes)
  32. Potassium oxalate (see Recipes)

Equipment

  1. Speedvac (Thermo Fisher Scientific, model: SPD131DDA )
  2. Thermomixer (Eppendorf)
  3. Sonicating Water Bath (Sigma-Aldrich, model: SONOREX Digital 10P )
  4. Mini-extruder set (Avanti, catalog number: 610000 )
  5. Hairdryer
  6. TLC chamber
  7. Fumehood
  8. Phosphoimager (optional) (Fujifilm, model: FLA-2000 )

Procedure

  1. Liposome preparation
    1. Lipids should be kept stored in a glass container in chloroform at -20 °C until required for use. Only prepare liposomes as required and use prepared liposomes within 24 h. Use a glass Hamilton syringe to transfer the required amount of phosphatidylinositol (PI) (10 mg/ml) in chloroform to a clean (methanol washed and dried) glass tube (Pyrex 12 x 75 mm). As 10 µg of extruded liposomes are used per reaction, it will require 1 µl per reaction to be carried out. It is advised to prepare slightly more liposomes than required as some volume is lost during the extrusion process (see step 1e). It is advisable to work with a minimum of 20 µl of chloroform (10 mg/ml) lipids; it is also advised to carry out each reaction in duplicate or triplicate (see Note 1 for further information).
    2. Evaporate the chloroform under a stream of nitrogen gas. Use a steady flow rate that is sufficient to disturb and push the lipid up against the side of the tube as it dries, this reduces the chances of residual chloroform becoming trapped under dried lipid. A subtle dried opaque coating should be visible on the tube.
    3. Dry the lipid further for 1 h in a speedvac with suitable attachment for the Pyrex tubes. Traces of chloroform can be detrimental to the assay, so it is important that all chloroform is evaporated.
    4. Re-suspend the PI in assay buffer (to give a 1 mg/ml solution) and allow 30 min to re-suspend with periodic vortexing and by using a sonicating water bath. The mixture should look chalky/cloudy. 
    5. Prepare the lipid mini-extruder by assembling with a 100 nm pore filter, assay buffer (~1 ml) can be used to flush through the extruder and ensure the system is assembled correctly without leakage and this will minimise any loss of volume. Detailed assembly instructions can be found via the Avanti website (http://avantilipids.com/divisions/equipment/mini-extruder-assembly-instructions/). Collect the lipid solution in one glass Hamilton syringe and attach to the extruder system. Pass slowly through without excessive pressure from one side to the other (~10 times) until the solution becomes clear (as seen on Avanti website: http://avantilipids.com/divisions/equipment/mini-extruder-extrusion-technique/), indicating that liposomes (Large unilamellar vesicles - LUVs) have been formed and are now in solution. Always ensure that the lipid mixture is at the opposite side to where it began so that everything has passed through the filter.
    6. The liposomes are now prepared and ready for use in the in vitro assay.
  2. In vitro phosphorylation of PI
    This assay is suitable for use with either recombinant (Bago et al., 2014) or immunoprecipitated VPS34 (Munson et al., 2015), although in the latter case it is important to ensure the antibody utilised does not interfere with activity. Follow a standard immunoprecipitation protocol to isolate VPS34 directly or co-immunoprecipitate it by targeting a binding partner (e.g., BECLIN1, ATG14L, UVRAG). As this assay involves the use of radioactive 32P-γ-ATP, ensure that all work is carried out in an area where radioactivity is allowed and meets local regulations and requirements for its use. When carrying out this assay, it is important to include appropriate controls such as a negative bead only/no kinase control, a positive kinase control [this could be recombinant protein of Class I or Class III PI3K as both will form PI(3)P in vitro from PI]. A PI3K inhibitor (such as Wortmannin or VPS34-IN1) can also be used to verify that PI(3)P formation is being observed (as these should block formation).
    1. Suspend either recombinant protein or beads if immunoprecipitating in a final volume of 40 μl assay buffer inside Eppendorf tubes. Dilute the extruded liposome mix within the 40 µl volume to a total amount of 10 μg of extruded liposomes per reaction. 
    2. Add 10 μl of the 5x ATP mix as a final addition to each sample and place directly into a thermomixer at 30 °C/1,100 rpm for 30 min. It is ideal to stagger the addition of ATP mix to each sample to allow time to process each sample at the end of the 30 min reaction. This time will vary dependent if the assay is being carried out on beads from an immunoprecipitation or with recombinant protein. It is advised to become familiar with the following steps and have a trial run to determine the optimal staggering time. 
    3. Terminate the reaction by the addition of 500 μl Stop solution. If using beads, then first separate the beads by passing through a Spin-X column before addition of Stop solution to the eluate. Sample buffer (1x) can be added to the beads retained in the column and re-eluted into a fresh tube. These samples can then be used for western blot to verify and determine the levels of immunoprecipitated protein between samples and serve as a control for the assay.
    4. Once all samples are in Stop solution, the components are phase-split by the addition of 180 μl chloroform and subsequently 300 μl 0.1 M HCl per sample. Vortex samples gently before centrifugation at 0.5 x g/1 min. The samples will split into an upper aqueous (water-methanol) and lower organic (chloroform-methanol) lipid-containing layer. 
    5. Prime a pipette tip by briefly pipetting chloroform up and down. Once primed, put the pipette tip through the methanol layer to aspirate the lower chloroform layer and remove to a separate tube. Expel the pipette whilst moving to collect the lower layer to minimise carry over. 
    6. Dry chloroform samples in a speedvac (~5-10 min). Once dry, samples can either be retained at -20 °C to run at a later date or used immediately for separation of lipid products by TLC.
  3. Thin layer chromatography (TLC)
    Lipid products can be separated by spotting and running on a Silica 60 TLC plate. A simple overview of the TLC process is shown in Figure 1.


    Figure 1. Overview of TLC procedure. Samples are spotted onto the origin (a point above the TLC solvent) and added to the TLC chamber containing solvent. As the solvent travels up the plate, the phosphorylated lipid products are separated. In this case, the lipid product PI(3)P cannot but seen by eye, but will be detected by the incorporation of 32P.

    1. Pre-treat the TLC plate by wetting completely in potassium oxalate solution to improve the resolution of phospholipids (Gonzalez-Sastre and Folch-Pi, 1968). Allow to dry fully, this can be left at RT overnight (~16 h) or can be dried more rapidly using a hairdryer. The TLC chamber should also be prepared in advance by addition of the TLC solvent to saturate the chamber. Whatman paper can be added at the sides of the chamber to improve speed and maintain even resolution when running the TLC plates.
    2. Mark gently in pencil where each sample is to be loaded at least 2 cm from the bottom of the TLC plate. The loaded samples must not be lower than the solvent level in the tank.
    3. Dried samples should be resuspended in 50-100 μl of chloroform and spotted onto the TLC plate. A glass narrow-bore capillary can be utilised for spotting samples onto the TLC plate. The sample should be applied gradually, allowing the chloroform to dry before adding further. Application speed can be improved by utilising a hairdryer to evaporate the chloroform quickly between spotting.
    4. Once completely loaded, the TLC plate should be placed into the TLC chamber and left until the solvent front reaches just below the top of the plate. The plate should then be removed and allowed to dry in the fume hood. 
      The level of phosphorylation can then be determined by multiple means. The incorporation of 32P correlates to phosphorylated lipid and can be measured by exposure to x-ray film. Other methods that have a greater linear range of detection such as a Phosphorimager can be utilised if available for more accurate quantitation. Assay of recombinant VPS34/VPS15 is very clean and produces a single band that migrates up the plate. Signal observed at the origin that does not migrate is likely to be carryover 32P-γ-ATP that has not been incorporated into a lipid product. 
      In the case of an immunoprecipitation, the retained sample buffer from step 2c can be analysed by western blot for VPS34 levels. This can then serve as a loading control to normalise the values obtained via the TLC plate.

Representative data



Figure 2. Example data from lipid kinase assay. Recombinant VPS34/VPS15 or Class I PI3Ka was assayed as described in the presence or absence of a pan PI3K inhibitor. Following loading of samples onto a TLC plate and running, the plate was exposed to x-ray film and developed. 

Notes

  1. The data obtained via lipid kinase assay is inherently more variable than a protein kinase assay, largely due to the greater variation in the substrate itself. Extruding lipids to form liposomes of maximal size 100 nm helps to make the substrate more uniform and reduce variability, although some will remain. Extruding the solution a second time will help to increase homogeneity further. Carrying out each reaction in duplicate or triplicate and taking the average value can also help to increase reliability. The level of homogeneity can be determined by performing dynamic light scattering (DLS).
  2. The curvature of liposomes may also affect the ability to act as a substrate for VPS34, certain sub-complexes are reported to have different phosphorylation kinetics dependent upon membrane curvature (Rostislavleva et al., 2015). It may therefore be wise also to consider use of different filter pore sizes when extruding to test a range of liposome sizes.
    Whilst assays of VPS34 activity can be very clean, it is prudent to include a suitable positive control to confirm that any bands observed via TLC are indeed PI(3)P. This could be either using recombinant VPS34/VPS15 or a class I PI3K [these will form PI(3)P from PI in vitro but not in cells]. Additionally, a specific VPS34 inhibitor such as VPS34-IN1 may be used to confirm any signal is VPS34 dependent (Bago et al., 2014).

Recipes

  1. ATP mix (5x)
    5 μCi 32P-γ-ATP
    5 μM ATP
    Prepare in kinase assay buffer
    Prepare fresh before use
  2. Kinase assay buffer
    20 mM Tris, pH 7
    67 mM NaCl
    10 mM MnCl2
    0.02% (w/v) CHAPS
    1 mM DTT
    Prepare fresh before use
  3. Stop solution
    Chloroform:methanol:12 M HCl (100:200:3.5, v/v)
    Store at RT in glass top bottles
    Be wary of chloroform evaporation over time
  4. Sample buffer (1x)
    62.5 mM Tris, pH 6.8
    10% (v/v) glycerol
    2% (w/v) SDS
    0.005% bromophenol blue
    2.5% (v/v) β-mercaptoethanol
    Can be stored at RT but add β-mercaptoethanol fresh
  5. TLC solvent
    Methanol:chloroform:water:14.5 M ammonium hydroxide (47:60:11.2:2, v/v)
    Store at RT in glass top bottles
    Solvent can be reused 2-3 times
    Be wary of chloroform evaporation over time
  6. Potassium oxalate
    1% (w/v) potassium oxalate
    5 mM EDTA
    50% (v/v) methanol
    Can be stored at RT

Acknowledgments

This work was supported by the Medical Research Council (MRC) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer).

References

  1. Bago, R., Malik, N., Munson, M. J., Prescott, A. R., Davies, P., Sommer, E., Shpiro, N., Ward, R., Cross, D., Ganley, I. G. and Alessi, D. R. (2014). Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase. Biochem J 463(3): 413-427.
  2. Devereaux, K., Dall'Armi, C., Alcazar-Roman, A., Ogasawara, Y., Zhou, X., Wang, F., Yamamoto, A., De Camilli, P. and Di Paolo, G. (2013). Regulation of mammalian autophagy by class II and III PI 3-kinases through PI3P synthesis. PLoS One 8(10): e76405.
  3. Gonzalez-Sastre, F. and Folch-Pi, J. (1968). Thin-layer chromatography of the phosphoinositides. J Lipid Res 9(4): 532-533.
  4. Munson, M. J., Allen, G. F., Toth, R., Campbell, D. G., Lucocq, J. M. and Ganley, I. G. (2015). mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival. EMBO J 34(17): 2272-2290.
  5. Rostislavleva, K., Soler, N., Ohashi, Y., Zhang, L., Pardon, E., Burke, J. E., Masson, G. R., Johnson, C., Steyaert, J., Ktistakis, N. T. and Williams, R. L. (2015). Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes. Science 350(6257): aac7365.
  6. Zhou, X., Takatoh, J. and Wang, F. (2011). The mammalian class 3 PI3K (PIK3C3) is required for early embryogenesis and cell proliferation. PLoS One 6(1): e16358.

简介

VPS34是哺乳动物细胞中唯一的III类磷脂酰肌醇-3-激酶(PI3K),并且产生绝大多数细胞磷脂酰肌醇-3-磷酸[PI(3)P]。 PI(3)P是一个关键的信号脂质,在诸如内吞作用和自噬的过程中起许多膜运输的作用。 VPS34是关键的细胞调节剂,功能丧失可具有灾难性作用并且是胚胎致死的(Zhou等人,2011)。 细胞PI(3)P的水平可以通过荧光染色技术确定,并且可以用于监测对VPS34活性的影响,然而重要的是验证任何变化由VPS34介导,特别是作为PI(3)的替代途径, P生产是可能的,例如通过II类PI3K(Devereaux等人,2013)。 直接在体外测定VPS34活性可以是描绘特定刺激的作用的关键阶段。

关键字:磷脂酰肌醇3 -磷酸, Vps34, pik3c3, 脂质激酶测定, 薄层色谱法

材料和试剂

  1. Hamilton注射器(500μl)(Hamilton,目录号:80865)
  2. Pyrex管(12×75mm)(Corning,目录号:99445-12)
  3. Eppendorf flex tube(Eppendorf,目录号:022364111)
  4. Spin-X柱(0.22μm)(Corning,Costar ,目录号:8161)
  5. Whatman纸(3mm)(Sigma-Aldrich,目录号:3030-917)
  6. 二氧化硅60 TLC板(Merck Millipore Corporation,目录号:1.05553.0001)
  7. TLC点样毛细管,15.2cm(VWR,目录号:80060-608)
  8. 聚碳酸酯膜,100nm孔径(Avanti Polar Lipids,目录号:610005)
  9. 氯仿(VWR International,目录号:22707.320)
  10. 粗制磷脂酰肌醇(PI)(Avanti,目录号:840042C)
  11. 甲醇(VWR International,目录号:20847.307)
  12. N 2气罐(99.998%纯)
  13. VPS34/VPS15重组蛋白(MRCPPU试剂,目录号:DU8692)
  14. 32-P-γ-ATP(PerkinElmer,目录号:NEG002A500UC)
  15. ATP(Sigma-Aldrich,目录号:A2383)
  16. 盐酸(37%)(VWR International,目录号:20252.335)
  17. 甘油(VWR International,目录号:24388.320)
  18. 十二烷基硫酸钠(SDS)(VWR International,目录号:444464T)
  19. 溴酚蓝(Sigma-Aldrich,目录号:114405)
  20. β-巯基乙醇
  21. Tris碱(VWR International,目录号:103157P)
  22. 氯化钠(VWR International,目录号:27810.364)
  23. 氯化锰(Sigma-Aldrich,目录号:M3634)
  24. CHAPS(Sigma-Aldrich,目录号:C3023)
  25. 二硫苏糖醇(DTT)(Formedium,目录号:DTT010)
  26. 氢氧化铵(Sigma-Aldrich,目录号:320145)
  27. ATP混合物(5x)(参见配方)
  28. 激酶测定缓冲液(参见配方)
  29. 停止解决方案(参见配方)
  30. 样品缓冲液(1x)(参见配方)
  31. TLC溶剂(见配方)
  32. 草酸钾(见配方)

设备

  1. Speedvac(Thermo Fisher Scientific,型号:SPD131DDA)
  2. 热固机(Eppendorf)
  3. 超声波水浴(Sigma-Aldrich,型号:SONOREX Digital 10P)
  4. 微型挤出机组(Avanti,目录号:610000)
  5. 吹风机
  6. TLC室
  7. 通风柜
  8. Phosphoimager(可选)(Fujifilm,型号:FLA-2000)

程序

  1. 脂质体制备
    1. 脂质应保存在-20℃的氯仿中的玻璃容器中,直到需要使用。仅根据需要制备脂质体,并在24小时内使用制备的脂质体。使用玻璃Hamilton注射器将所需量的氯仿中的磷脂酰肌醇(PI)(10mg/ml)转移到干净的(甲醇洗涤并干燥的)玻璃管(Pyrex 12×75mm)。由于每次反应使用10μg的挤出脂质体,因此每次反应需要1μl。建议准备略多于所需的脂质体,因为在挤出过程中一些体积损失(参见步骤1e)。建议最少使用20μl氯仿(10 mg/ml)脂质,建议每次反应一式两份,一式三份(更多信息,请参见注1)。
    2. 在氮气流下蒸发氯仿。使用稳定的流速,其在干燥时足以干扰和推动脂质向管的侧面,这降低了残留的氯仿被捕获在干脂质下的可能性。微妙的干燥的不透明涂层应该在管上可见。
    3. 干燥脂质进一步1小时在speedvac与适当的附件Pyrex管。氯仿的痕量可能对测定有害,因此重要的是所有氯仿被蒸发。
    4. 将PI在测定缓冲液中重悬(得到1mg/ml溶液),并允许30分钟通过定期涡旋和通过使用超声处理水浴重新悬浮。混合物应该看起来白垩/多云。
    5. 通过与100nm孔过滤器组装来制备脂质微型挤出机,可以使用测定缓冲液(〜1ml)冲洗通过挤出机,并确保系统正确组装,没有泄漏,这将最小化任何体积损失。详细的组装说明可通过Avanti网站( http://avantilipids.com/divisions/equipment/mini-extruder-assembly-instructions/)。收集脂质溶液在一个玻璃Hamilton注射器,并附加到挤出机系统。慢慢地通过,没有过多的压力从一侧到另一侧(〜10次),直到解决方案变清楚(如在Avanti网站上看到的: http://avantilipids.com/divisions/equipment/mini-extruder-extrusion-technique/),表明脂质体(大单层囊泡 - LUVs)已经形成并且现在在溶液中。始终确保脂质混合物在开始的相对侧,以便一切都已经通过过滤器。
    6. 现在制备脂质体并准备好用于体外试验。
  2. PI的体外磷酸化
    该测定法适用于重组(Bago等人,2014)或免疫沉淀的VPS34(Munson等人,2015),尽管在后一种情况下,它是重要的是确保所使用的抗体不干扰活性。遵循标准免疫沉淀方案直接分离VPS34或通过靶向结合配偶体(例如,BECLIN1,ATG14L,UVRAG)进行共免疫沉淀。由于该测定涉及使用放射性β32 P-γ-ATP,确保所有工作在允许放射性的区域进行,并且符合当地法规和使用要求。当进行该测定时,重要的是包括 适当的对照例如仅负珠/无激酶对照,阳性激酶对照[这可以是I类或III类PI3K的重组蛋白,因为二者将在体外从PI形成PI(3)P ]。 PI3K抑制剂(例如渥曼青霉素或VPS34-IN1)也可用于验证PI(3)P形成被观察到(因为它们应该阻断形成)。
    1. 如果在Eppendorf管内的终体积为40μl测定缓冲液中免疫沉淀,则悬浮重组蛋白或珠。在40μl体积内稀释挤出的脂质体混合物至每次反应的总量为10μg的挤出脂质体。
    2. 加入10μl的5x ATP混合物作为每个样品的最后添加,并且以30℃/1,100rpm直接放入热混合器中30分钟。理想的是,将ATP混合物加入到每个样品中以允许时间在30分钟反应结束时处理每个样品。如果在来自免疫沉淀的珠子上或用重组蛋白质进行测定,则该时间将变化。建议熟悉以下步骤,并试运行以确定最佳的交错时间。
    3. 通过加入500μl终止溶液终止反应。如果使用珠,则首先通过通过Spin-X柱分离珠,然后将终止液加入洗脱液中。可以将样品缓冲液(1x)加入保留在柱中的珠中,并重新洗脱到新管中。然后这些样品可用于蛋白质印迹以验证和确定样品之间免疫沉淀的蛋白质的水平,并用作测定的对照。
    4. 一旦所有样品都在停止溶液中,通过加入180μl氯仿并随后加入每个样品300μl0.1M HCl来对组分进行相分离。在0.5离心之前轻轻涡旋样品 x g /1分钟。样品将分裂成上层水(水 - 甲醇)和下层有机(氯仿 - 甲醇)含脂层。
    5. 通过简单地用氯仿上下移动吸移移液管吸头。一旦准备好,将移液器吸头通过甲醇层吸出下面的氯仿层,并移除到一个单独的管。排出移液器,同时移动以收集较低的层,以尽量减少结转。
    6. 在speedvac中干燥氯仿样品(〜5-10分钟)。一旦干燥,样品可以保存在-20℃以在以后运行或立即使用通过TLC分离脂质产物。
  3. 薄层色谱(TLC)
    脂质产物可以通过点样并在硅胶60 TLC板上运行来分离。 TLC过程的简单概述如图1所示。

    ="">
    图1. TLC过程概述将样品点在原点(TLC溶剂上方的点)上,并加入到含有溶剂的TLC室中。当溶剂沿着板向上移动时,磷酸化的脂质产物被分离。在这种情况下,脂质产物PI(3)P不能仅仅通过肉眼看到,而是通过掺入 P来检测。

    1. 通过在草酸钾溶液中完全润湿来预处理TLC板,以提高磷脂的分辨率(Gonzalez-Sastre和Folch-Pi,1968)。使其完全干燥,可以在室温下放置过夜(〜16小时),或者可以使用吹风机更快地干燥。 TLC室也应该预先通过加入TLC溶剂以使室饱和而制备。 Whatman纸可以添加在腔的侧面,以提高速度和运行TLC板时保持均匀的分辨率。
    2. 在铅笔上轻轻地标记,其中每个样品要从TLC板的底部至少2cm加载。装载的样品不得低于罐中的溶剂水平。
    3. 干燥样品应重悬于50-100μl氯仿中,并点样到TLC板上。可以使用玻璃窄孔毛细管将样品点样到TLC板上。应该逐渐施加样品,在进一步加入之前使氯仿干燥。通过使用吹风机在点样之间快速蒸发氯仿可以提高施用速度。
    4. 一旦完全装载,TLC板应该被放置到TLC室中并且留下直到溶剂前沿刚好到达板的顶部下方。然后应该取出板,并在通风橱中干燥。
      然后可以通过多种方法测定磷酸化水平。 32的掺入与磷酸化脂质相关,并且可以通过暴露于x射线胶片来测量。如果可用于更准确的定量,则可以利用具有更大线性范围的检测的其他方法,例如磷光成像仪。重组VPS34/VPS15的测定非常干净,并产生在板上迁移的单一条带。在原点观察到的信号 不迁移可能是未结合到脂质产物中的携带 P-γ-ATP。
      在免疫沉淀的情况下,来自步骤2c的保留的样品缓冲液可以通过蛋白质印迹分析VPS34水平。 这可以用作加载控制,以便对通过TLC板获得的值进行归一化

代表数据



图2.来自脂质激酶测定的实施例数据如在存在或不存在pan PI3K抑制剂的条件下所述测定重组VPS34/VPS15或I类PI3Kα。 将样品装载到TLC板上并运行后,将板暴露于X-射线胶片并显影。

笔记

  1. 通过脂质激酶测定获得的数据本质上比蛋白激酶测定更可变,主要是由于底物本身的更大变化。挤出脂质以形成最大尺寸100nm的脂质体有助于使底物更均匀和减少变异性,尽管一些将保留。第二次挤出溶液将有助于进一步提高均匀性。进行每个反应一式两份或三次,并取平均值也可以有助于提高可靠性。均匀性水平可以通过进行动态光散射(DLS)来测定。
  2. 脂质体的曲率也可以影响作为VPS34的底物的能力,据报道某些亚复合物具有取决于膜弯曲的不同的磷酸化动力学(Rostislavleva等人,2015)。因此,当挤出以测试一系列脂质体大小时,考虑使用不同的过滤器孔径也是明智的。
    尽管VPS34活性的测定可以非常干净,但是谨慎地包括合适的阳性对照以证实通过TLC观察到的任何条带确实是PI(3)P。这可以是使用重组VPS34/VPS15或I类PI3K [这些将在体外形成PI(3)P,但不在细胞中形成PI(3)P]。另外,特定的VPS34抑制剂例如VPS34-IN1可用于确认任何信号是VPS34依赖性的(Bago等人,2014)。

食谱

  1. ATP混合物(5x)
    5μCi<32> P-γ-ATP 5μMATP
    在激酶测定缓冲液中制备
    使用前准备新鲜
  2. 激酶测定缓冲液
    20mM Tris,pH7
    67 mM NaCl 10mM MnCl 2
    0.02%(w/v)CHAPS
    1 mM DTT
    使用前准备新鲜
  3. 停止解决方案
    氯仿:甲醇:12M HCl(100:200:3.5,v/v)洗脱 在室温下储存在玻璃瓶中
    注意随着时间的推移氯仿蒸发
  4. 示例缓冲区(1x)
    62.5mM Tris,pH 6.8
    10%(v/v)甘油 2%(w/v)SDS
    0.005%溴酚蓝
    2.5%(v/v)β-巯基乙醇 可以在室温下储存,但加入β-巯基乙醇新鲜
  5. TLC溶剂
    甲醇:氯仿:水:14.5M氢氧化铵(47:60:11.2:2,v/v) 在室温下储存在玻璃瓶中
    溶剂可重复使用2-3次
    注意随着时间的推移氯仿蒸发
  6. 草酸钾
    1%(w/v)草酸钾 5 mM EDTA
    50%(v/v)甲醇
    可以在RT存储

致谢

这项工作由医学研究理事会(MRC)和支持信号转导治疗单位(AstraZeneca,Boehringer-Ingelheim,GlaxoSmithKline,Merck KGaA,Janssen Pharmaceutica和Pfizer)的制药公司支持。

参考文献

  1. Bago,R.,Malik,N.,Munson,MJ,Prescott,AR,Davies,P.,Sommer,E.,Shpiro,N.,Ward,R.,Cross,D.,Ganley,IGand Alessi,DR (2014)。 VPS34-IN1,Vps34的选择性抑制剂的表征揭示磷脂酰肌醇3-磷酸结合SGK3蛋白激酶是III类磷酸肌醇3-激酶的下游靶。 Biochem J 463(3):413-427。
  2. Devereaux,K.,Dall'Armi,C.,Alcazar-Roman,A.,Ogasawara,Y.,Zhou,X.,Wang,F.,Yamamoto,A.,De Camilli,P.and Di Paolo, (2013年)。 通过II类和III类PI 3激酶调节哺乳动物自噬PI3P合成。 PLoS One 8(10):e76405。
  3. Gonzalez-Sastre,F。和Folch-Pi,J。(1968)。 磷酸肌醇的薄层色谱。 J Lipid Res 9(4):532-533。
  4. Munson,M.J.,Allen,G.F.,Toth,R.,Campbell,D.G.,Lucocq,J.M。和Ganley,I.G。(2015)。 mTOR激活VPS34-UVRAG复合物以调节自体溶酶体的细胞和细胞存活。 EMBO J 34(17):2272-2290。
  5. Rostislavleva,K.,Soler,N.,Ohashi,Y.,Zhang,L.,Pardon,E.,Burke,JE,Masson,GR,Johnson,C.,Steyaert,J.,Ktistakis, (2015)。 内体Vps34复合物的结构和灵活性揭示了其功能的基础在膜上。 科学 350(6257):aac7365。
  6. Zhou,X.,Takatoh,J.和Wang,F。(2011)。 哺乳类3类PI3K(PIK3C3)是早期胚胎发生所必需的, 细胞增殖。 PLoS One 6(1):e16358
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Munson, M. J. and Ganley, I. G. (2016). Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP. Bio-protocol 6(16): e1904. DOI: 10.21769/BioProtoc.1904.
  2. Munson, M. J., Allen, G. F., Toth, R., Campbell, D. G., Lucocq, J. M. and Ganley, I. G. (2015). mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival. EMBO J 34(17): 2272-2290.
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