5蛋白激酶与磷酸酶ppt课件

1、蛋白激酶与磷酸酶蛋白激酶与磷酸酶梅柱中梅柱中病理生理学教研室病理生理学教研室 蛋白质翻译后修饰蛋白质翻译后修饰Post-translational modifications (PTMs) 磷酸化修饰Phosphorylation ） 糖基化修饰Glycosylation ） 泛素化修饰Ubiquitination ） 乙酰化修饰Acetylation ） 甲基化修饰Methylation ） SUMOylation磷酸化修饰的氨基酸磷酸化修饰的氨基酸蛋白质的磷酸化修饰蛋白质的磷酸化修饰导致受体蛋白的激活或失活；2% 蛋白质的磷酸化修饰发生在Tyr残基上；大部分的早期信号转导是由酪氨酸蛋白激酶

2、介导的protein tyrosine kinase, PTKs)；Ser/Thr磷酸化修饰多发生于信号转导级联反应的下游。蛋白激酶将ATP末端的磷酸基团转移到蛋白质侧链的羟基上；磷酸酶催化蛋白质侧链上磷酸基团的水解。ProteinOH + ATPProteinOPOOO+ ADPPiH2O Protein Kinase Protein Phosphatase 蛋白激酶与磷酸酶蛋白激酶与磷酸酶 30%的蛋白质可以发生磷酸化修饰的蛋白质可以发生磷酸化修饰 人类的激酶组包括人类的激酶组包括518蛋白激酶基因蛋白激酶基因 其中其中218个基因与人类疾病的发生发展密切相关个基因与人类疾病的发生发展密切

3、相关 约约30个基因是肿瘤抑制基因个基因是肿瘤抑制基因 约约100个为原癌基因个为原癌基因Human Kinome 约占人类基因总数的约占人类基因总数的1.7%蛋白激酶的结构蛋白激酶的结构激酶结构域Kinase domain）调节结构域Regulatory domain）靶向性结构域Targeting domain）蛋白激酶的分类蛋白激酶的分类 酪氨酸蛋白激酶 丝氨酸/苏氨酸蛋白激酶 (PKC,MAPK,Plk,Rho Kinases) 双特异性蛋白激酶 (MAPKK) 磷酯酰肌醇激酶 ( PI3K)受体激酶 (EGFR,FGFR,PDGFR)非受体激酶 (JAK,Src,Abl)受体酪氨酸激

4、酶受体酪氨酸激酶RTK）受体酪氨酸激酶包括大多数生长因子的受体，参与了细胞的多种生命活动如细胞的生长、细胞形态与细胞周期调控、基因的转录与细胞凋亡等；RTKs通常是含有单次跨膜结构域的单体蛋白分子，与配基结合后发生二聚体化，使细胞内的结构域靠近，通过自磷酸化而激活； 该家族受体的激活与许多原癌基因的活化密切相关。Six subfamilies of receptor tyrosine kinases involved in cell growth and differentiation. Only one or two members of each subfamily are indicat

5、ed. Note that the tyrosine kinase domain is interrupted by a kinase insert region in some of the subfamilies. The functional significance of the cysteine-rich and immunoglobulin-like domains is unknown. 受体酪氨酸激酶受体酪氨酸激酶RTK）受体二聚体化与激酶的激活受体二聚体化与激酶的激活Binding of insulin to the alpha subunits causes the bet

6、a subunits to phosphorylate themselves (autophosphorylation), thus activating the catalytic activity of the receptor. 胰岛素受体胰岛素受体MAPKK可同时催化MAPK激酶的T与Y的磷酸化修饰： 磷酸化位点的三肽模体 TXY ERK TEY p38 TGY JNK TPYMAPK是通过Thr和Tyr的双位点同时磷酸化而被激活。双特异性蛋白激酶双特异性蛋白激酶磷酸肌醇循环磷酸肌醇循环PIPI-PhosphataseooooOHOHHOHOPPIPPI-Kinasephosphati

7、dylinositolphosphoinositidesooooOHHOPP345PPHO磷酸肌醇循环磷酸肌醇循环PI3K-PKB/Akt信号通路Western blot analysis of whole cell lysates of Jurkat cells, with 0.1M calyculin A for 20 minutes prior to lysis, using Phospho-Thr antibody. + PPP inhibitor蛋白激酶与磷酸酶蛋白激酶与磷酸酶 丝氨酸/苏氨酸蛋白磷酸酶 (PP1, PP2A)； 酪氨酸蛋白磷酸酶 (PTP1B)； 双特异性蛋白磷酸酶

8、 (DUSP1)； 脂质磷酸酶 (PTEN, SHIP)。蛋白质磷酸酶蛋白质磷酸酶Ser/Thr与与Tyr蛋白磷酸酶蛋白磷酸酶催化机制：催化机制：含有双核的金属离子中心含有双核的金属离子中心；通过水分子来进行亲核攻通过水分子来进行亲核攻击；击；磷酸基团不转移到磷酸酶磷酸基团不转移到磷酸酶上，是一步催化反应。上，是一步催化反应。结构上：结构上：数量有限的催化亚基与众数量有限的催化亚基与众多调节亚基配对。多调节亚基配对。 没有金属离子的参与； 通过催化结构域的半胱氨酸残基进行亲核攻击； 形成磷酸基-酶中间体，是二步催化反应。 多基因家族，在单一磷酸酶中同时含有催化与调节结构域。Ser/Thr磷酸酶

9、 (PP1)Tyr磷酸酶 (PTP, DUSP1)Fe O O H O P O-Ser-substrateZn OH-HisSer/Thr蛋白磷酸酶催化模式图蛋白磷酸酶催化模式图PP2A磷酸酶的多样性与复杂性磷酸酶的多样性与复杂性翻译后修饰对磷酸酶功能的调控作用翻译后修饰对磷酸酶功能的调控作用磷酸化修饰的级联反应磷酸化修饰的级联反应Signal moleculeActiveproteinkinase1Activeproteinkinase2Activeproteinkinase3Inactiveprotein kinase1Inactiveprotein kinase2Inactiveprot

10、ein kinase3InactiveproteinActiveproteinCellularresponseReceptorPPP PPPATPADPADPADPATPATPPPPPPPActivated relaymolecule A relay moleculeactivates protein kinase 1.1 Active protein kinase 1transfers a phosphate from ATPto an inactive molecule ofprotein kinase 2, thus activatingthis second kinase.2 Acti

11、ve protein kinase 2then catalyzes the phos-phorylation (and activation) ofprotein kinase 3.3 Finally, active proteinkinase 3 phosphorylates aprotein (pink) that brings about the cells response tothe signal.4 Enzymes called proteinphosphatases (PP)catalyze the removal ofthe phosphate groupsfrom the p

12、roteins, making them inactiveand available for reuse.5iiiPhosphorylation cascadeMAPK信号级联反应信号级联反应StimulusGrowth factors, Mitogen, GPCRp38 MAPKStress, GPCR, Inflammatory cytokines, Growth factorsStress, Growth factors, Mitogen, GPCRMEKK1, 4, MLK3, ASK1MEKK2, 3, Tpl2MLK3, TAK, DLKRaf, Mos, Tpl2MKK3/6ME

13、K1/2MKK4/7MEK5ERK1/2MAPKKKGrowth, Differentiation, DevelopmentInflammation, Apoptosis, Growth, DifferentiationGrowth, Differentiation, DevelopmentERK5/BMK1JNK1,2,3MAPKKMAPKBiological responses磷酸酶的信号级联反应磷酸酶的信号级联反应S287S287T138T13814-3-314-3-3During interphase, Cdc25 is held inactive via by inhibitory

14、phosphorylation at Ser287 and 14-3-3 binding. Likewise, PP2A/B56 maintains Thr138 in the dephosphorylated state. At the G2/M transition, Cdc25 is activated in a stepwise fashion. First, Cdk2 phosphorylates Thr138 which triggers the release of 14-3-3. Phosphorylated keratin intermediate filaments ass

15、ist in 14-3-3 removal from Cdc25 and Plx1 may also play a role in this process. Exposed Ser287 is then readily dephosphorylated by PP1, inducing the activation and nuclear translocation of Cdc25 and dephosphorylation of Cdc2. Once activated, Cdc2/Cyclin B phosphorylates multiple sites on Cdc25, enha

16、ncing its activity and preventing inactivation. Cdc2/Cyclin B may also activate the MAP kinase cascade which can phosphorylate Cdc25 in a parallel positive feedback loop. 位点特异性的磷酸化修饰位点特异性的磷酸化修饰Kinase: ATM, ATR, DNAPK, CK1, CK2, Chk1, Chk2, etc.p53:蛋白质的磷酸化修饰分析蛋白质的磷酸化修饰分析生物质谱分析蛋白质的磷酸化修饰生物质谱分析蛋白质的磷酸化修饰EEVAS*EPEEAAS*PTTPKEEVAS*EPEEAASPTTPKEEVASEPEEAASPTTPKy1-y5, My6-y12, M+80y13-y17,M+160y1-y17, My1-y12, My13-y17,M+80IMAC: Immobilized Metal Affinity Chromatography 固定化金属离子亲和性层析法SIMAC: sequential elution fr