蛋白激酶与磷酸酶 病理生理学教研室

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

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。

蛋白激酶与磷酸酶 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。

人类的激酶组包括518蛋白激酶基因 约占人类基因总数的1.7% 其中218个基因与人类疾病的发生发展密切相关 约30个基因是肿瘤抑制基因 Human Kinome 人类的激酶组包括518蛋白激酶基因 约占人类基因总数的1.7% 其中218个基因与人类疾病的发生发展密切相关 约30个基因是肿瘤抑制基因 约100个为原癌基因 ~30%的蛋白质可以发生磷酸化修饰

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 酪氨酸蛋白激酶 受体激酶 (EGFR,FGFR,PDGFR) 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。

受体酪氨酸激酶（RTK） 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 "kinase insert region" in some of the subfamilies. The functional significance of the cysteine-rich and immunoglobulin-like domains is unknown.

受体二聚体化与激酶的激活

胰岛素受体 Binding of insulin to the alpha subunits causes the beta subunits to phosphorylate themselves (autophosphorylation), thus activating the catalytic activity of the receptor.

双特异性蛋白激酶 MAPK是通过Thr和Tyr的双位点同时磷酸化而被激活。 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 — TPY MAPK是通过Thr和Tyr的双位点同时磷酸化而被激活。

磷酸肌醇循环 PI-Kinase PI PIP PI-Phosphatase phosphatidylinositol phosphoinositides PI-Kinase HO OH 4 3 P P HO OH P OH HO 5 P HO P o o PI PIP o o o o o o PI-Phosphatase

磷酸肌醇循环

PI3K-PKB/Akt信号通路 GF: such as EGF, TGF, PDGF etc. Mutated ras 17

蛋白激酶与磷酸酶 + PPP inhibitor 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.

蛋白质磷酸酶 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 (DUSP1)； 脂质磷酸酶 (PTEN, SHIP)。

Ser/Thr与Tyr蛋白磷酸酶 Ser/Thr磷酸酶 (PP1) Tyr磷酸酶 (PTP, DUSP1) 催化机制： 结构上： 含有双核的金属离子中心； 通过水分子来进行亲核攻击； 磷酸基团不转移到磷酸酶上，是一步催化反应。 结构上： 数量有限的催化亚基与众多调节亚基配对。 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蛋白磷酸酶催化模式图 Fe O O H O P O-Ser-substrate Zn O H-His

PP2A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 Schematic representation of c-Src activation at the onset of mitosis. (A) During most of the cell cycle, c-Src is largely phosphorylated at Tyr527 and therefore in an inactive tail-bite conformation, whereas 20% of RPTP is phosphorylated at Tyr789 and saturated with Grb2. (B) At the transition from G2 to M phase, c-Src is first phosphorylated in its NH2-terminus by p34cdc2, and RPTP is phosphorylated at several serines by an unknown kinase, perhaps PKC. Subsequently, Grb2 dissociates from RPTP . (C) c-Src binds through its SH2 domain to phospho-Tyr789 on RPTP . This binding event exposes phospho-Tyr527 for dephosphorylation by the D1 domain of RPTP , allowing c-Src to autophosphorylate at Tyr416, thereby becoming fully active. PTK, protein tyrosine kinase.

activates protein kinase 1.  磷酸化修饰的级联反应 Signal molecule Receptor Activated relay molecule A relay molecule activates protein kinase 1. 1 Inactive protein kinase 1 Active protein kinase 1 transfers a phosphate from ATP to an inactive molecule of protein kinase 2, thus activating this second kinase. 2 Active protein kinase 1 Phosphorylation cascade Inactive protein kinase 2 ATP ADP Active protein kinase 2 P Active protein kinase 2 then catalyzes the phos- phorylation (and activation) of protein kinase 3. 3 PP P i Inactive protein kinase 3 ATP ADP Active protein kinase 3 P Finally, active protein kinase 3 phosphorylates a protein (pink) that brings about the cell’s response to the signal. 4 Enzymes called protein phosphatases (PP) catalyze the removal of the phosphate groups from the proteins, making them inactive and available for reuse. 5 PP P i Inactive protein ATP ADP P Active protein Cellular response PP P  i

MAPK信号级联反应 Stimulus MAPKKK MAPKK MAPK Biological responses Stress, GPCR, Inflammatory cytokines, Growth factors Stress, Growth factors, Mitogen, GPCR Growth factors, Mitogen, GPCR Stimulus Raf, Mos, Tpl2 MEKK1, 4, MLK3, ASK1 MEKK2, 3, Tpl2 MAPKKK MLK3, TAK, DLK MAPKK MEK1/2 MKK3/6 MKK4/7 MEK5 MAPK ERK1/2 p38 MAPK JNK1,2,3 ERK5/BMK1 Growth, Differentiation, Development Inflammation, Apoptosis, Growth, Differentiation Growth, Differentiation, Development Biological responses

 磷酸酶的信号级联反应 14-3-3 S287 14-3-3 T138 S287 T138

During interphase, Cdc25 is held inactive via by inhibitory phosphorylation at Ser287 and 14-3-3 binding. 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. Phosphorylated 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 translocation 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 cascade which can phosphorylate Cdc25 in a parallel positive feedback loop.

位点特异性的磷酸化修饰 p53: T55/S376:在正常细胞中发生磷酸化修饰，但在受到应激刺激时发生去磷酸化； Kinase: ATM, ATR, DNAPK, CK1, CK2, Chk1, Chk2, etc. T55/S376:在正常细胞中发生磷酸化修饰，但在受到应激刺激时发生去磷酸化； S15/S20: 磷酸化修饰降低其与Hdm2的结合，并促进与转录共刺激分子的结合，但它们在细胞中的功能是冗余的； S46：磷酸化修饰可诱导其下游的促凋亡蛋白基因如p53AIP1等的表达； S392: UV诱导其磷酸化修饰，其功能与抑制UV诱导的皮肤癌密切相关。

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 EEVASEPEEAASPTTPK y1-y17, M EEVAS*EPEEAASPTTPK

磷酸化蛋白质组（Phosphoproteome） IMAC: Immobilized Metal Affinity Chromatography 固定化金属离子亲和性层析法 SIMAC: sequential elution from IMAC 从IMAC顺序洗脱

DUSP1

PI3K信号通路与癌症治疗 PI3K信号通路参与调控细胞的生长、存活、代谢等重要的生命活动； 其特异性的磷酸酶PTEN是癌细胞中最常发生突变的抑癌基因之一，仅次于p53； PI3K激酶上下游分子如受体酪氨酸激酶、Akt激酶等的激活也可以促进癌症的发生与发展。

PI3K信号通路与癌症治疗 1. Wortmannin is a reactive electrophilic and therefore unstable compound whereas LY294002 is a weak inhibitor with only micromolar potency, and both agents display significant off-target effects on other kinases。 39