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PKC-θ in vitro Kinase Activity Assay
PKC-θ激酶体外活性试验   

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Abstract

Protein kinase C-θ (PKC-θ), a member of the Ca2+-independent PKC subfamily of kinases, serves as a regulator of T cell activation by mediating the T cell antigen receptor (TCR)- and coreceptor CD28-induced activation of the transcription factors NF-κB and AP-1 and, to a lesser extent, NFAT, and, subsequently, interleukin 2 (IL-2) production and T cell proliferation. In T cells, TCR and CD28 stimulation-induced activation of PKC-θ is the integrated result of diacylglycerol-mediated membrane recruitment, GLK-mediated phosphorylation at activation loop, CD28, Lck, and sumoylation-mediated central immunological synapse localization (Wang et al., 2015; Monks et al., 1997; Kong et al., 2011; Isakov and Altman, 2012; Chuang et al., 2011). Phosphatidylserine (PtdSer) and the phorbol ester Phorbol 12-myristate 13-acetate (PMA, a surrogate of diacylglycerol [DAG]) are the cofactors for the Ca2+-independent PKC subfamily that bind to PKC directly and activate it by changing its conformation (Nishizuka, 1995). A protocol to analyze the PKC-θ kinase activity in vitro is described here. Myelin basic protein is used as the substrate and its phosphorylation is detected by the incorporation of radioactive phosphate into the substrate, which is analyzed by a laser scanner.

Keywords: In vitro kinase assay(体外激酶测定), PKC-θ(PKC), Radiolabeled ATP(放射性标记的ATP)

Background

Specified by their divergent regulatory domains, the PKC family can be divided into distinct subgroups: the conventional PKCs (cPKCs, comprising PKC-α, β and γ), the novel PKCs (nPKCs, including PKC-δ, ε, θ and η) and the atypical PKCs (aPKCs, PKCι and ζ). The cPKCs are activated by a combination of diacylglycerol, phospholipid and Ca2+. The nPKCs are activated by diacylglycerol and phorbol esters but do not require Ca2+. In contrast, the aPKCs do not depend on Ca2+ or diacylglycerol for activation (Rosse et al., 2010). Here we used anti-Myc or anti-PKC-θ to isolate PKC-θ from the cell lysates, then performed the radiolabeled ATP based kinase assay in the Reaction buffer containing PMA, PtdSer and EGTA (a selective chelator for Ca2+). The assay protocol described here is quick, sensitive and specific, provides a direct measurement of PKC-θ activity. This protocol could be modified to analyze the activity of other nPKC isoforms.

Materials and Reagents

  1. Jurkat E6.1 cells or HEK293T cells
  2. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  3. RPMI-1640 medium (GE Healthcare, HyCloneTM, catalog number: SH30027.01 )
  4. Heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  5. Anti-PKC-θ (Santa Cruz Biotechnology, catalog number: sc-1875 )
  6. Anti-Myc (9E10) (Santa Cruz Biotechnology, catalog number: sc-40 )
  7. Protein G sepharose (GE Healthcare, catalog number: 17-0618-01 )
  8. Phosphatidylserine from PepTag® Non-Radioactive PKC assay kit (Promega, catalog number: V5330 )
  9. Tris (Sangon Biotech, catalog number: A600194 )
  10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  11. Ethylenediaminetetraacetate (EDTA) (Sangon Biotech, catalog number: A610185 )
  12. Nonidet P40 (Sangon Biotech, catalog number: A600385 )
  13. Aprotinin (EMD Millipore, catalog number: 616370 )
  14. Leupeptin (Calbiochem, catalog number: 108976 )
  15. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  16. Sodium pyrophosphate tetrabasic decahydrate (NaPPi) (Sigma-Aldrich, catalog number: S6422 )
  17. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: 567540 )
  18. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 11344041 )
  19. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M4880 )
  20. EGTA (Sigma-Aldrich, catalog number: E3889 )
  21. ATP (Sigma-Aldrich, catalog number: A2383 )
  22. [γ-32P]ATP (PerkinElmer, catalog number: NEG002Z001MC )
  23. Myelin basic protein (MBP) (Sigma-Aldrich, catalog number: M1891 )
  24. PMA (Sigma-Aldrich, catalog number: P1585 )
  25. Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
  26. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
  27. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  28. Glycerol (Sangon Biotech, catalog number: A100854 )
  29. ATP-competitive PKC-θ inhibitor rottlerin (EMD Millipore, catalog number: 557370 )
  30. DMSO
  31. Lysis buffer (see Recipes)
  32. PKC-θ kinase buffer (see Recipes)
  33. Reaction buffer (see Recipes)
  34. 5x SDS-PAGE loading buffer (see Recipes)

Equipment

  1. Typhoon Trio+ system (GE Healthcare)
  2. Humidified CO2 incubator
  3. Laminar air flow bio-safety cabinet
  4. Centrifuge (Eppendorf, model: 5417R )
  5. Phosphor-storage screen (GE Healthcare)
  6. Sonicator (Sonics, model: VCX-130 )
  7. Thermomixer (Eppendorf)
  8. Exposure cassettes (GE Healthcare)

Software

  1. Typhoon Scanner Control software

Procedure

  1. Harvest 5 x 106 Jurkat E6.1 cells or 1 x 106 HEK293T cells transfected with vector, wild type (WT), kinase-dead mutant (K409R) and sumoylation deficient mutant (2KR [K325R, K506R]) of Myc-PKC-θ (indicated in Figure 2) by centrifugation (300 x g, 3 min, 25 °C).
  2. Wash the cells once with 1 ml ice cold PBS.
  3. Centrifuge the cells at 300 x g for 3 min at 25 °C.
  4. Add 300-500 µl lysis buffer to the cell pellets, vortex for 1-2 min.
  5. Place the tube on ice for 10 min.
  6. Centrifuge at 8,500 x g for 10 min at 4 °C.
  7. Transfer the clear supernatant to a new microcentrifuge tube.
  8. Immunoprecipitate endogenous or transfected PKC-θ by adding 2.5 µl anti-PKC-θ or anti-Myc antibodies into the clear supernatant obtained in step 7. Incubate overnight at 4 °C with rotation. Then add 30 µl protein G sepharose beads (50% [v/v] in PBS) and incubate for 2-4 h at 4 °C with rotation.
  9. Centrifuge for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant. Wash the immunoprecipitates extensively by adding 1 ml lysis buffer and vortexing for 3-5 min.
  10. Repeat step 9.
  11. Centrifuge for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant. Wash the immunoprecipitates extensively by adding 1 ml PKC-θ kinase buffer and vortexing for 3-5 min.
  12. Repeat step 11.
  13. Centrifuge for 5 min at 8,500 x g at 4 °C, carefully remove the supernatant and resuspend the pellet in 25 μl of the reaction buffer.
  14. Incubate samples in the Thermomixer with gentle shaking at 300 rpm for 30 min at 30 °C. Stop the reaction by adding 6.25 µl 5x SDS-PAGE loading buffer.
  15. Heat samples at 95 °C for 10 min and subject the samples to 15% SDS-PAGE, then cover the gel with plastic wrap to minimize the chance of radioactive contamination.
  16. Open the exposure cassette, place the wrapped gel on the internal surface of the cassette, and then place the phosphor-storage screen on top the wrapped gel with the phosphor (white) side facing down onto the gel. Close and lock the cassette, place it for 30 min to 6 h at room temperature and then take the phosphor-storage screen out of the cassette avoiding direct light. Scan the screen with the Typhoon Trio+ system.
  17. (Optional) A pilot experiment to verify the specificity of this kinase assay is recommended. In detail, HEK293T cells were transfected with wild type Myc-PKC-θ, which has proven to be an active kinase as T538, S676, and S695 are constitutively phosphorylated on recombinant PKC-θ isolated from HEK293T expression systems (Wang et al., 2012). 24 h after transfection, Myc-PKC-θ was immunoprecipitated and subjected to the kinase assay as described previously. When doing the kinase assay, the ATP-competitive PKC-θ inhibitor or DMSO was added into the reaction buffer which served as the negative and the positive control individually.

Data analysis

  1. The kinase assay system works specifically if there is a strong signal for the DMSO added sample while no signal for the PKC-θ inhibitor added sample in the pilot experiment.
  2. Analyze signals with Typhoon Scanner Control software as followed:
    1. Double-click the Scanner Control shortcut icon on the desktop.
    2. After the current instrument state is ‘Ready’, select the acquisition mode as ‘Storage Phosphor’ (Figure 1).


      Figure 1. The scanner control window

    3. Place the storage phosphor screen in the Typhoon instrument. Remove the storage phosphor screen from the exposure cassette. Keep the screen face down. Position the screen on the glass platen so that the A1 corner of the screen aligns with the A1 corner of the glass platen. Close the sample lid.
    4. Select the grid area and pixel size, click ‘SCAN’ to start scanning.
    5. Save the data and evaluate the results. A representative data set for the samples from transfected HEK293T cells was shown in Figure 2.

Representative data



Figure 2. In vitro kinase assay of Myc-tagged PKC-θ. Myc-PKC-θ-WT, -2KR (K325R, K506R) or -K409R were immunoprecipitated (IP) from lysates of HEK293T cells transfected with empty vector or the indicated Myc-tagged PKC-θ plasmids, MBP was used as a substrate in the presence of PtdSer/PMA cofactors (upper panel). Aliquots of the IPs were immunoblotted with anti-Myc to confirm similar expression levels of PKC-θ (bottom panel).

Notes

Phosphatidylserine easily aggregates to form micelles. To achieve maximal PKC-θ activation, sonicate the phosphatidylserine solution with a probe sonicator for 20-30 sec or until warm to disrupt the micelles.

Recipes

  1. Lysis buffer
    20 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    5 mM EDTA
    1% Nonidet P40
    10 μg/ml aprotinin
    10 μg/ml leupeptin
    1 mM PMSF
    5 mM NaPPi
    1 mM Na3VO4
  2. PKC-θ kinase buffer
    20 mM HEPES
    10 mM MgCl2
    0.1 mM EGTA
  3. Reaction buffer
    5 μCi [γ-32P]ATP, 20 μM ATP
    1 μg of myelin basic protein (MBP)
    10 μM PMA cofactors
    200 μg/ml phosphatidylserine (PtdSer) (presence or absence)
  4. 5x SDS-PAGE loading buffer
    0.25% bromophenol blue
    0.5 M DTT
    50% glycerol
    10% SDS
    0.25 M Tris-HCl (pH 6.8)

Acknowledgments

Supported by the National Natural Science Foundation of China (31170846) and the Ministry of Science and Technology of China (2013CB835300). This protocol was modified from the in vitro kinase assay (Bi, 2001).

References

  1. Bi, K., Tanaka, Y., Coudronniere, N., Sugie, K., Hong, S., van Stipdonk, M. J. and Altman, A. (2001). Antigen-induced translocation of PKC-θ to membrane rafts is required for T cell activation. Nat Immunol 2(6): 556-563.
  2. Chuang, H. C., Lan, J. L., Chen, D. Y., Yang, C. Y., Chen, Y. M., Li, J. P., Huang, C. Y., Liu, P. E., Wang, X. and Tan, T. H. (2011). The kinase GLK controls autoimmunity and NF-κB signaling by activating the kinase PKC-θ in T cells. Nat Immunol 12(11): 1113-1118.
  3. Isakov, N. and Altman, A. (2012). PKC-theta-mediated signal delivery from the TCR/CD28 surface receptors. Front Immunol 3: 273.
  4. Kong, K. F., Yokosuka, T., Canonigo-Balancio, A. J., Isakov, N., Saito, T. and Altman, A. (2011). A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28. Nat Immunol 12(11): 1105-1112.
  5. Monks, C. R., Kupfer, H., Tamir, I., Barlow, A. and Kupfer, A. (1997). Selective modulation of protein kinase C-θ during T-cell activation. Nature 385(6611): 83-86.
  6. Nishizuka, Y. (1995). Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9(7): 484-496.
  7. Rosse, C., Linch, M., Kermorgant, S., Cameron, A. J., Boeckeler, K. and Parker, P. J. (2010). PKC and the control of localized signal dynamics. Nat Rev Mol Cell Biol 11(2): 103-112.
  8. Xie, J. J., Liang, J. Q., Diao, L. H., Altman, A. and Li, Y. (2013). TNFR-associated factor 6 regulates TCR signaling via interaction with and modification of LAT adapter. J Immunol 190(8): 4027-4036.
  9. Wang, X., Chuang, H. C., Li, J. P. and Tan, T. H. (2012). Regulation of PKC-θ function by phosphorylation in T cell receptor signaling. Front Immunol 3: 197.
  10. Wang, X. D., Gong, Y., Chen, Z. L., Gong, B. N., Xie, J. J., Zhong, C. Q., Wang, Q. L., Diao, L. H., Xu, A., Han, J., Altman, A. and Li, Y. (2015). TCR-induced sumoylation of the kinase PKC-θ controls T cell synapse organization and T cell activation. Nat Immunol 16(11): 1195-1203.

简介

Sumoylation控制许多细胞过程。蛋白激酶C-θ(PKC-θ)是激酶的Ca 2+超家族PKC亚家族的成员,通过介导T细胞抗原受体(TCR)作为T细胞活化的调节剂, - 和共受体CD28诱导的转录因子NF-κB和AP-1的活化,以及较小程度的NFAT,以及随后的白细胞介素2(IL-2)产生和T细胞增殖。我们最近证明了TCR诱导的PKC-θ的sumoylation是其在T细胞中的功能所必需的(Wang等人,2015)。在这里我们描述分析TCR诱导的过表达或内源性PKC-θ的sumoylation的方法,这是通过免疫沉淀PKC-θ进行,然后用抗SUMO1抗体进行免疫印迹。

[背景] 与泛素化一样,sumoylation是用SUMO共价修饰目标蛋白的过程。为了破坏非共价相互作用并且特异性地检测目标蛋白上的sumoylation,应当使用用于细胞裂解,免疫沉淀和洗涤的严格条件。然而,用含有1%或更多SDS的裂解缓冲液裂解细胞产生高粘性细胞裂解物,使得难以进行免疫沉淀和免疫印迹步骤。在这里我们描述了一个替代的lysing-denaturing过程。首先,将细胞在补充有SUMO特异性蛋白酶抑制剂N,N-乙基马来酰亚胺的裂解缓冲液中裂解。离心后,将1%SDS加入到细胞裂解物的上清液中。将裂解物用补充有N-乙基马来酰亚胺的裂解缓冲液稀释10倍,并进行免疫沉淀。该方案也可以用于检测其他泛素样修饰,如Neddylation。...

关键字:体外激酶测定, PKC, 放射性标记的ATP

材料和试剂

  1. Jurkat E6.1细胞或HEK293T细胞
  2. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  3. RPMI-1640培养基(GE Healthcare,HyClone ,目录号:SH30027.01)
  4. 热灭活的胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
  5. 抗PKC-θ(Santa Cruz Biotechnology,目录号:sc-1875)
  6. 抗-Myc(9E10)(Santa Cruz Biotechnology,目录号:sc-40)
  7. 蛋白G琼脂糖凝胶(GE Healthcare,目录号:17-0618-01)
  8. 来自PepTag 非放射性PKC测定试剂盒(Promega,目录号:V5330)的磷脂酰丝氨酸
  9. Tris(Sangon Biotech,目录号:A600194)
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  11. 乙二胺四乙酸(EDTA)(Sangon Biotech,目录号:A610185)
  12. Nonidet P40(Sangon Biotech,目录号:A600385)
  13. 抑肽酶(EMD Millipore,目录号:616370)
  14. 亮肽素(Calbiochem,目录号:108976)
  15. 苯基甲磺酰基吡咯(PMSF)(Sigma-Aldrich,目录号:P7626)
  16. 焦磷酸四钠十水合物(NaPPi)(Sigma-Aldrich,目录号:S6422)
  17. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:567540)
  18. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:11344041)
  19. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M4880)
  20. EGTA(Sigma-Aldrich,目录号:E3889)
  21. ATP(Sigma-Aldrich,目录号:A2383)
  22. [γ-32 P] ATP(PerkinElmer,目录号:NEG002Z001MC)
  23. 髓磷脂碱性蛋白(MBP)(Sigma-Aldrich,目录号:M1891)
  24. PMA(Sigma-Aldrich,目录号:P1585)
  25. 溴酚蓝(Bio-Rad,目录号:1610404)
  26. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
  27. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  28. 甘油(Sangon Biotech,目录号:A100854)
  29. ATP竞争性PKC-θ抑制剂rottlerin(EMD Millipore,目录号:557370)
  30. DMSO
  31. 裂解缓冲液(见配方)
  32. PKC-θ激酶缓冲液(参见配方)
  33. 反应缓冲液(参见配方)
  34. 5x SDS-PAGE上样缓冲液(见配方)

设备

  1. Typhoon Trio + 系统(GE Healthcare)
  2. 加湿CO 2培养箱
  3. 层流气流生物安全柜
  4. 离心机(Eppendorf,型号:5417R)
  5. 荧光屏(GE Healthcare)
  6. 超声波仪(Sonics,型号:VCX-130)
  7. 热固机(Eppendorf)
  8. 曝光盒(GE Healthcare)

软件

  1. 台风扫描仪控制软件

程序

  1. 收获5×10 6个用载体,野生型(WT),激酶失活突变体(K409R)和苏氨酰化转染的Jurkat E6.1细胞或1×10 6个HEK293T细胞通过离心(300×g,3分钟,25℃)分离Myc-PKC-θ(图2所示)的缺陷型突变体(2KR [K325R,K506R])。
  2. 用1ml冰冷的PBS洗涤细胞一次。
  3. 在25℃下以300×g离心细胞3分钟。
  4. 向细胞沉淀中加入300-500μl裂解缓冲液,涡旋1-2分钟
  5. 将管在冰上放置10分钟。
  6. 在4℃下以8,500×g离心10分钟。
  7. 将清澈的上清液转移到新的微量离心管中
  8. 通过向步骤7中获得的澄清上清液中加入2.5μl抗PKC-θ或抗Myc抗体来免疫沉淀内源性或转染的PKC-θ。在4℃下旋转过夜孵育。然后加入30μl蛋白G琼脂糖珠(50%[v/v]的PBS溶液),并在4℃下旋转孵育2-4小时。
  9. 在4℃下以8,500×g离心5分钟,小心地除去上清液。通过加入1ml裂解缓冲液并涡旋3-5分钟来广泛洗涤免疫沉淀物
  10. 重复步骤9.
  11. 在4℃下以8,500×g离心5分钟,小心地除去上清液。通过加入1ml PKC-θ激酶缓冲液并涡旋3-5分钟来广泛地洗涤免疫沉淀物。
  12. 重复步骤11.
  13. 在4℃下以8,500×g离心5分钟,小心地除去上清液并将沉淀重悬于25μl反应缓冲液中。
  14. 孵育样品在Thermomixer中轻轻摇动300 rpm在30℃30分钟。通过加入6.25μl5x SDS-PAGE上样缓冲液停止反应。
  15. 在95℃下加热样品10分钟,并将样品进行15%SDS-PAGE,然后用塑料包膜覆盖凝胶,以最小化放射性污染的机会。
  16. 打开曝光盒,将包裹的凝胶放置在盒的内表面上,然后将荧光粉存储屏放置在包裹的凝胶上,使荧光体(白色)面朝下到凝胶上。关闭并锁定磁带,将其在室温下放置30分钟至6小时,然后将荧光粉存储屏幕从磁带盒中取出,避免直接照明。使用Typhoon Trio + 系统扫描屏幕。
  17. (可选)建议进行试验性实验以验证该激酶测定的特异性。详细地,用已经证明是活性激酶的野生型Myc-PKC-θ转染HEK293T细胞,因为T538,S676和S695在从HEK293T表达系统分离的重组PKC-θ上组成型磷酸化(Wang等人al 。,2012)。转染后24小时,Myc-PKC-θ免疫沉淀,并如前所述进行激酶测定。当进行激酶测定时,将ATP竞争性PKC-θ抑制剂或DMSO加入到单独作为阴性和阳性对照的反应缓冲液中。

数据分析

  1. 如果对于加入的DMSO样品存在强信号,则激酶测定系统具体工作,而在试验实验中没有PKC-θ抑制剂样品的信号。
  2. 用Typhoon Scanner Control软件分析信号如下:
    1. 双击桌面上的"扫描仪控制"快捷方式图标。
    2. 当前仪器状态为"Ready"后,选择采集模式为"存储荧光粉"(图1)。


      图1.扫描仪控制窗口

    3. 将存储荧光屏放在Typhoon仪器中。从曝光盒上取下存储荧光屏。保持屏幕正面朝下。将屏幕放置在玻璃压板上,使屏幕的A1角与玻璃压板的A1角对齐。关闭样品盖。
    4. 选择网格区域和像素大小,单击"扫描"开始扫描。
    5. 保存数据并评估结果。来自转染的HEK293T细胞的样品的代表性数据显示于图2中。

代表数据



图2. Myc标记的PKC-θ的体外激酶测定。 Myc-PKC-θ-WT,-2KR(K325R,K506R)或-K409R免疫沉淀IP)从用空载体或指定的Myc标记的PKC-θ质粒转染的HEK293T细胞的裂解物中,在PtdSer/PMA辅因子(上图)存在下,MBP用作底物。用抗-Myc免疫印迹IP的等分试样以确认PKC-θ的类似表达水平(下图)。

笔记

磷脂酰丝氨酸容易聚集形成胶束。为了实现最大PKC-θ活化,用探针超声波仪超声处理磷脂酰丝氨酸溶液20-30秒或直到温热以破坏胶束。

食谱

  1. 裂解缓冲液
    20mM Tris-HCl(pH7.5) 150mM NaCl 5 mM EDTA
    1%Nonidet P40
    10μg/ml抑肽酶
    10μg/ml亮肽素 1mM PMSF
    5 mM NaPPi
    1mM Na 3 VO 4 sub。
  2. PKC-θ激酶缓冲液
    20 mM HEPES
    10mM MgCl 2/
    0.1 mM EGTA
  3. 反应缓冲液
    5μCi[γ-32 P] ATP,20μMATP 1μg髓磷脂碱性蛋白(MBP)
    10μMPMA辅因子
    200μg/ml磷脂酰丝氨酸(PtdSer)(存在或不存在)
  4. 5x SDS-PAGE上样缓冲液
    0.25%溴酚蓝
    0.5 M DTT
    50%甘油 10%SDS
    0.25M Tris-HCl(pH 6.8)

致谢

由中国国家自然科学基金(31170846)和中国科学技术部支持(2013CB835300)。该方案从体外激酶测定(Bi,2001)修改。

参考文献

  1. Bi,K.,Tanaka,Y.,Coudronniere,N.,Sugie,K.,Hong,S.,van Stipdonk,MJ and Altman,A。(2001)。  抗原诱导的PKC-θ向膜筏的易位是T细胞活化所必需的。 > Nat Immunol 2(6):556-563
  2. (a,b,c,c,d,c,d,c,d,c,d,d) "ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21983831"target ="_ blank">激酶GLK通过激活激酶PKC-θ来控制自身免疫和NF-κB信号传导T cells。 Nat Immunol 12(11):1113-1118。
  3. Isakov,N。和Altman,A。(2012)。  PKC-θ介导的来自TCR/CD28表面受体的信号传递。前免疫 3:273.
  4. 在这种情况下,我们可以使用一个简单的例子来说明这个问题,即:激酶PKC-θ的V3结构域中的基序通过与CD28的结合确定其在免疫突触中的定位和在T细胞中的功能。 Nat Immunol 12(11):1105-1112。
  5. Monks,CR,Kupfer,H.,Tamir,I.,Barlow,A.和Kupfer,A。(1997)。  蛋白激酶C和脂质信号传导持续的细胞反应。 FASEB J 9(7):484-496。
  6. Rosse,C.,Linch,M.,Kermorgant,S.,Cameron,AJ,Boeckeler,K.and Parker,PJ(2010)。  通过磷酸化在T细胞受体信号传导中调节PKC-θ功能。前免疫 3:197.
  7. Wang,XD,Gong,Y.,Chen,ZL,Gong,BN,Xie,JJ,Zhong,CQ,Wang,QL,Diao,LH,Xu,A.,Han,J.,Altman, Y.(2015)。  TCR诱导的激酶PKC-θ控制T细胞突触组织和T细胞活化。自身免疫 16(11):1195-1203。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, X. and Li, Y. (2016). PKC-θ in vitro Kinase Activity Assay. Bio-protocol 6(20): e1980. DOI: 10.21769/BioProtoc.1980.
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