蛋白激酶與磷酸酶

1、蛋白激酶與磷酸酶,病理生理學(xué)教研室,蛋白質(zhì)翻譯后修飾,Post-translational modifications (PTMs) 磷酸化修飾（Phosphorylation ） 糖基化修飾（Glycosylation ） 泛素化修飾（Ubiquitination ） 乙?；揎棧ˋcetylation ） 甲基化修飾（Methylation ） SUMOylation……,磷酸化修飾的氨基酸,蛋白質(zhì)的磷酸化修飾,導(dǎo)致受體

2、蛋白的激活或失活；<2% 蛋白質(zhì)的磷酸化修飾發(fā)生在Tyr殘基上；大部分的早期信號(hào)轉(zhuǎn)導(dǎo)是由酪氨酸蛋白激酶介導(dǎo)的（protein tyrosine kinase, PTKs)；Ser/Thr磷酸化修飾多發(fā)生于信號(hào)轉(zhuǎn)導(dǎo)級(jí)聯(lián)反應(yīng)的下游。,蛋白激酶將ATP末端的磷酸基團(tuán)轉(zhuǎn)移到蛋白質(zhì)側(cè)鏈的羥基上；磷酸酶催化蛋白質(zhì)側(cè)鏈上磷酸基團(tuán)的水解。,蛋白激酶與磷酸酶,~30%的蛋白質(zhì)可以發(fā)生磷酸化修飾,人類的激酶組包括518蛋白激酶基因,其中218

3、個(gè)基因與人類疾病的發(fā)生發(fā)展密切相關(guān),約30個(gè)基因是腫瘤抑制基因,約100個(gè)為原癌基因,Human Kinome,約占人類基因總數(shù)的1.7%,蛋白激酶的結(jié)構(gòu),激酶結(jié)構(gòu)域（Kinase domain）調(diào)節(jié)結(jié)構(gòu)域（Regulatory domain）靶向性結(jié)構(gòu)域（Targeting domain）,蛋白激酶的分類,酪氨酸蛋白激酶 絲氨酸/蘇氨酸蛋白激酶 (PKC,MAPK,Plk,Rho Kinases) 雙特異性蛋白激酶 (MAPK

4、K) 磷酯酰肌醇激酶 ( PI3K),受體激酶 (EGFR,FGFR,PDGFR),非受體激酶 (JAK,Src,Abl),,受體酪氨酸激酶（RTK）,受體酪氨酸激酶包括大多數(shù)生長(zhǎng)因子的受體，參與了細(xì)胞的多種生命活動(dòng)如細(xì)胞的生長(zhǎng)、細(xì)胞形態(tài)與細(xì)胞周期調(diào)控、基因的轉(zhuǎn)錄與細(xì)胞凋亡等；RTKs通常是含有單次跨膜結(jié)構(gòu)域的單體蛋白分子，與配基結(jié)合后發(fā)生二聚體化，使細(xì)胞內(nèi)的結(jié)構(gòu)域靠近，通過自磷酸化而激活； 該家族受體的激活與許多原癌基因的活化

5、密切相關(guān)。,Six subfamilies of receptor tyrosine kinases involved in cell growth and differentiation. Only one or two members of each subfamily are indicated. Note that the tyrosine kinase domain is interrupted by a "kina

6、se insert region" in some of the subfamilies. The functional significance of the cysteine-rich and immunoglobulin-like domains is unknown.,,受體酪氨酸激酶（RTK）,受體二聚體化與激酶的激活,Binding of insulin to the alpha subunits causes t

7、he beta subunits to phosphorylate themselves (autophosphorylation), thus activating the catalytic activity of the receptor.,胰島素受體,MAPKK可同時(shí)催化MAPK激酶的T與Y的磷酸化修飾： 磷酸化位點(diǎn)的三肽模體 — TXY ERK — TEY p38 — TGY

8、 JNK — TPYMAPK是通過Thr和Tyr的雙位點(diǎn)同時(shí)磷酸化而被激活。,雙特異性蛋白激酶,磷酸肌醇循環(huán),PI,,,,,,,,,,,,,,,,,,,,,,,,,,,,,o,o,o,o,,OH,OH,HO,HO,P,PIP,phosphatidylinositol,phosphoinositides,,,,,,,,,,,,,o,o,o,o,,,,,,,,,,,,,,,,,,OH,HO,P,3,4,5,HO,磷酸肌醇循環(huán),

9、,,,,,PI3K-PKB/Akt信號(hào)通路,Western blot analysis of whole cell lysates of Jurkat cells, with 0.1?M calyculin A for 20 minutes prior to lysis, using Phospho-Thr antibody.,+ PPP inhibitor,蛋白激酶與磷酸酶,絲氨酸/蘇氨酸蛋白磷酸酶 (PP1, PP2A)； 酪氨酸

10、蛋白磷酸酶 (PTP1B)； 雙特異性蛋白磷酸酶 (DUSP1)； 脂質(zhì)磷酸酶 (PTEN, SHIP)。,蛋白質(zhì)磷酸酶,Ser/Thr與Tyr蛋白磷酸酶,催化機(jī)制：含有雙核的金屬離子中心；通過水分子來進(jìn)行親核攻擊；磷酸基團(tuán)不轉(zhuǎn)移到磷酸酶上，是一步催化反應(yīng)。結(jié)構(gòu)上：數(shù)量有限的催化亞基與眾多調(diào)節(jié)亞基配對(duì)。,沒有金屬離子的參與；通過催化結(jié)構(gòu)域的半胱氨酸殘基進(jìn)行親核攻擊； 形成磷酸基-酶中間體，是二步催化反應(yīng)。多基因家

11、族，在單一磷酸酶中同時(shí)含有催化與調(diào)節(jié)結(jié)構(gòu)域。,Ser/Thr磷酸酶 (PP1),Tyr磷酸酶 (PTP, DUSP1),,,,,Fe O O H O P O-Ser-substrateZn O,,,,,,,,H-His,,,,Ser/Thr蛋白磷酸酶催化模式圖,PP2A磷酸酶的多樣性與復(fù)雜性,翻譯后修飾對(duì)磷酸酶功能的

12、調(diào)控作用,磷酸化修飾的級(jí)聯(lián)反應(yīng),Signal molecule,Activeproteinkinase1,Activeproteinkinase2,Activeproteinkinase3,Inactiveprotein kinase1,Inactiveprotein kinase2,Inactiveprotein kinase3,Inactiveprotein,Activeprotein,,Cellu

13、larresponse,Receptor,,P,P,P,P,P,P,ATP,ADP,ADP,ADP,ATP,ATP,PP,PP,PP,Activated relaymolecule,,Active protein kinase 1transfers a phosphate from ATPto an inactive molecule ofprotein kinase 2, thus activatingthis secon

14、d kinase.,,,,,,,,,,,,i,i,i,,Phosphorylation cascade,MAPK信號(hào)級(jí)聯(lián)反應(yīng),,,,,,,,,,,,,Stimulus,Growth factors, Mitogen, GPCR,p38 MAPK,Stress, GPCR, Inflammatory cytokines, Growth factors,Stress, Growth factors, Mitogen, GPCR,MEKK1

15、, 4, MLK3, ASK1,MEKK2, 3, Tpl2,MLK3, TAK, DLK,Raf, Mos, Tpl2,MKK3/6,MEK1/2,MKK4/7,MEK5,ERK1/2,MAPKKK,Growth, Differentiation, Development,Inflammation, Apoptosis, Growth, Differentiation,Growth, Differentiation, Developm

16、ent,ERK5/BMK1,JNK1,2,3,MAPKK,MAPK,Biological responses,,,,,,,,,,,,,,,,,,,,,磷酸酶的信號(hào)級(jí)聯(lián)反應(yīng),S287,S287,T138,T138,14-3-3,14-3-3,During interphase, Cdc25 is held inactive via by inhibitory phosphorylation at Ser287 and 14-3-3 bin

17、ding. Likewise, PP2A/B56δ 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. Phosphor

18、ylated keratin intermediate filaments assist 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 translo

19、cation of Cdc25 and dephosphorylation of Cdc2. Once activated, Cdc2/Cyclin B phosphorylates multiple sites on Cdc25, enhancing its activity and preventing inactivation. Cdc2/Cyclin B may also activate the MAP kinase casc

20、ade which can phosphorylate Cdc25 in a parallel positive feedback loop.,位點(diǎn)特異性的磷酸化修飾,Kinase: ATM, ATR, DNAPK, CK1, CK2, Chk1, Chk2, etc.,T55/S376:在正常細(xì)胞中發(fā)生磷酸化修飾，但在受到應(yīng)激刺激時(shí)發(fā)生去磷酸化；S15/S20: 磷酸化修飾降低其與Hdm2的結(jié)合，并促進(jìn)與轉(zhuǎn)錄共刺激分子的結(jié)合，但它們

21、在細(xì)胞中的功能是冗余的；S46：磷酸化修飾可誘導(dǎo)其下游的促凋亡蛋白基因如p53AIP1等的表達(dá)；S392: UV誘導(dǎo)其磷酸化修飾，其功能與抑制UV誘導(dǎo)的皮膚癌密切相關(guān)。,p53:,蛋白質(zhì)的磷酸化修飾分析,生物質(zhì)譜分析蛋白質(zhì)的磷酸化修飾,EEVAS*EPEEAAS*PTTPK,EEVAS*EPEEAASPTTPK,EEVASEPEEAASPTTPK,y1-y5, My6-y12, M+80y13-y17,M+160,y1-y17,

22、 M,y1-y12, My13-y17,M+80,生物質(zhì)譜分析蛋白質(zhì)的磷酸化修飾,IMAC: Immobilized Metal Affinity Chromatography 固定化金屬離子親和性層析法SIMAC: sequential elution from IMAC 從IMAC順序洗脫,磷酸化蛋白質(zhì)組（Phosphoproteome）,DUSP1,PI3K信號(hào)通路與癌癥治療,PI3K信號(hào)通路參與調(diào)控細(xì)胞的生長(zhǎng)、存活、代謝等