功能蛋白的O-糖基化和p38磷酸化共同参与调控单核细胞对血管内皮的粘附和侵袭

探讨单核细胞在炎症因子刺激下通过功能蛋白O-糖基化和p38 MAPK磷酸化、调控其对血管内皮的粘附和侵袭的分子机制。将IFN-γ与LPS体外共刺激后的THP-1细胞加至单层血管内皮细胞EA.hy926共培养,观察单核细胞对血管内皮的粘附和侵袭;并通过测量电阻变化来反应血管内皮通透性的改变。采用Western blot方法检测单核细胞THP-1中p38 MAPK磷酸化的变化,O-GLcNAc糖基转移酶（OGT）和O-GLcNAc糖基化蛋白表达量的变化。分析验证p38 MAPK抑制剂对IFN-γ与LPS诱导的单核细胞对血管内皮粘附和迁移的影响,同时检测OGT、O-GLcNAc糖基化蛋白差异表达的影响。结果显示,IFN-γ与LPS可以共作用促进THP-1对血管内皮的粘附和侵袭,降低血管内皮通透性。同时激活p38 MAPK,此过程与OGT及O-GLcNAc糖基化蛋白表达降低相关。采用p38抑制剂预处理,可逆转上述IFN-γ与LPS诱导的生物学变化。综上,在炎症反应中,单核细胞对血管内皮的粘附和侵袭力的变化受功能蛋白糖基化和磷酸化的双向调控。     

p38 MAPK通路在脂多糖致呼吸道上皮损伤中的作用

脂多糖（Lipopolysaccharide,LPS）是革兰阴性杆菌细胞壁的主要组成成分,也是一种很强的炎症反应和氧化应激诱导剂。呼吸道上皮是机体防御外界细菌、病毒、香烟烟雾等生物和化学因素损伤的天然屏障,在维持呼吸道局部微环境稳态中可发挥重要作用,也是吸入性药物治疗的主要靶细胞。呼吸道上皮结构完整性缺陷或功能紊乱还参与了哮喘、慢性阻塞性肺疾病等多种肺部疾病的发生和发展。LPS可引起呼吸道上皮损伤,但其具体的分子机制目前尚不清楚。p38丝裂原活化蛋白激酶（P38mitogen-activated protein kinase,p38 MAPK）作为MAPK家族四个亚家族成员之一,包含四个成员：p38α、p38β、p38γ和p38δ,可通过经典和非经典的p38 MAPK信号通路激活方式及通过激酶活性无关的功能参与调控炎症反应、细胞生长、细胞分化和细胞死亡等多种病理生理过程。本文就p38 MAPK信号通路在LPS致呼吸道上皮损伤中的作用做一综述。     

CCK-8对内毒素休克大鼠肺脏细胞因子的抑制效应

观察八肽胆囊收缩素(cholecystokinin-octapeptide,CCK-8）改善脂多糖(lipopolysaccharide,LPS）引起的大鼠内毒素性休克（endotoxic shock,ES）过程中血清及肺脏细胞因子的变化,探讨p38比裂素活化蛋白激酶（p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入LPS(p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入 LPS（8mg/kg i.v.）复制的SD大鼠ES模型、LPS注入前10min尾静脉注入CCK-8(40ug/kg i.v.）、单独注入CCK-8(40Uug/kg i.v.）或生理盐水（对照）的四组大鼠平均动脉血压（MAP）的改变,应用ELISA试剂盒检测血清和肺脏中炎性细胞因子（TNF-a、IL-1β和IL-6）的变化。用Western blot检测肺脏p38 MAPK的表达。结果显示：CCK-8可改善LPS引起的大鼠MAP的下降。与对照组相比,LPS可显著增加血清和肺脏TNF-a、IL-1β和IL-6含量;CCK-8可显著抑制LPS诱导的血清和肺脏TNF-a、IL-1β和IL-6的增加。CCK-8可增加ES大鼠肺脏磷酸化p38 MAPK的表达。结果提示CCK-8可改善ES大鼠MAP的降低,并对肺脏促炎性细胞因子过量产生有抑制作用,p38MAPK可能参与了其信号转导机制。     

木犀草素通过抑制p65 NF-κB、促进p85 PI3K调节微血管内皮细胞VCAM-1表达（英文）

通过抑制微血管内皮细胞血管细胞黏附分子(VCAM)-1的表达,木犀草素可阻遏中性粒细胞与微血管内皮细胞的黏附,起到抗炎作用.木犀草素调节VCAM-1表达与三条信号通路有关:丝裂原活化蛋白激酶(MAPK)、核因子kappa B(NF-κB)/IκB和磷脂酰肌醇3激酶(PI3K)/Akt通路.其中,MAPK和NF-κB/IκB通路参与VCAM-1正向调节,PI3K/Akt通路参与VCAM-1负向调节.本文研究了木犀草素对微血管内皮细胞该三条通路中的关键蛋白p38 MAPK、p65 NF-κB、p85 PI3K磷酸化.结果表明:木犀草素在反应的30 s和1 min促进p38 MAPK磷酸化,在30 s、1 min和5 min促进p85 PI3K磷酸化,而在30 s、1 min、5 min和30 min抑制p65 NF-κB磷酸化.阻抑p38 MAPK通路导致VCAM-1表达下调,而p38 MAPK抑制剂SB203580可通过抑制p38 MAPK磷酸化也下调VCAM-1,提示木犀草素对微血管内皮细胞VCAM-1的调节作用独立于p38 MAPK磷酸化.由此可知,木犀草素通过抑制p65 NF-κB磷酸化或促进p85 PI3K磷酸化调节微血管内皮细胞VCAM-1表达.本文为木犀草素抗炎作用的分子机制研究提供了新的线索.     

p38信号通路参与LPS诱导的Raw264.7细胞骨架重构

应用免疫荧光标记、基因转染等方法,观察脂多糖（LPS）刺激对单核细胞系Raw264.7细胞骨架的影响,探讨p38家族不同亚型对LPS诱导的细胞骨架蛋白微管蛋白与肌动蛋白变化的调控作用结果显示,未受LPS刺激的细胞富含微管蛋白,微管蛋白交联形成辐射状的交联丝网,丝网在细胞中分布均匀;LPS刺激后,微管蛋白募集在细胞膜、核膜周围;p38α、p38β、p38γ亚型的特异性抑制剂FHPI对LPS诱导的微管蛋白募集无影响,而p38无活性突变体p38δ(AF)的基因转染,可抑制LPS诱导的细胞骨架微管蛋白的募集;肌动蛋白在静息的细胞内主要存在于细胞膜周围,LPS作用后,肌动蛋白在细胞中形成广泛分布的辐射状应激纤维;p38上游激酶活性诱变体MKK6b基因转染可诱导Raw细胞形成类似的应激纤维,而p38γ(AF)的基因转染,可抑制LPS诱导的细胞应激纤维的形成.上述结果表明,p38δ可能参与了LPS诱导的微管蛋白的重构;而LPS诱导Raw细胞应激纤维的形成,可能是通过p38γ蛋白激酶而发挥作用.     

P38在LPS诱导大鼠雪旺氏(Schwann) 细胞TNF-α表达中的作用

应用免疫荧光标记、基因转染等方法,观察脂多糖（LPS）刺激对单核细胞系Raw264.7细胞骨架的影响,探讨p38家族不同亚型对LPS诱导的细胞骨架蛋白微管蛋白与肌动蛋白变化的调控作用结果显示,未受LPS刺激的细胞富含微管蛋白,微管蛋白交联形成辐射状的交联丝网,丝网在细胞中分布均匀;LPS刺激后,微管蛋白募集在细胞膜、核膜周围;p38α、p38β、p38γ亚型的特异性抑制剂FHPI对LPS诱导的微管蛋白募集无影响,而p38无活性突变体p38δ(AF)的基因转染,可抑制LPS诱导的细胞骨架微管蛋白的募集;肌动蛋白在静息的细胞内主要存在于细胞膜周围,LPS作用后,肌动蛋白在细胞中形成广泛分布的辐射状应激纤维;p38上游激酶活性诱变体MKK6b基因转染可诱导Raw细胞形成类似的应激纤维,而p38γ(AF)的基因转染,可抑制LPS诱导的细胞应激纤维的形成.上述结果表明,p38δ可能参与了LPS诱导的微管蛋白的重构;而LPS诱导Raw细胞应激纤维的形成,可能是通过p38γ蛋白激酶而发挥作用.     

木犀草素通过抑制p65 NF-κB、促进p85 PI3K调节微血管内皮细胞VCAM-1表达

通过抑制微血管内皮细胞血管细胞黏附分子(VCAM)-1的表达,木犀草素可阻遏中性粒细胞与微血管内皮细胞的黏附,起到抗炎作用。木犀草素调节VCAM-1表达与三条信号通路有关：丝裂原活化蛋白激酶(MAPK)、核因子kappa B (NF-κB)/IκB和磷脂酰肌醇3激酶(PI3K)/Akt通路。其中,MAPK和NF-κB/IκB通路参与VCAM-1正向调节,PI3K/Akt通路参与VCAM-1负向调节。本文研究了木犀草素对微血管内皮细胞该三条通路中的关键蛋白p38 MAPK、p65 NF-κB、p85 PI3K磷酸化。结果表明：木犀草素在反应的30s和1min促进p38 MAPK磷酸化,在30 s、1 min和5 min促进p85 PI3K磷酸化,而在30 s、1 min、5 min和30 min抑制p65 NF-κB磷酸化。阻抑p38 MAPK通路导致VCAM-1表达下调,而p38 MAPK抑制剂SB203580可通过抑制p38 MAPK磷酸化也下调VCAM-1,提示木犀草素对微血管内皮细胞VCAM-1的调节作用独立于p38 MAPK磷酸化。由此可知,木犀草素通过抑制p65 NF-κB磷酸化或促进p85 PI3K磷酸化调节微血管内皮细胞VCAM-1表达。本文为木犀草素抗炎作用的分子机制研究提供了新的线索。     

姜黄素对内毒素诱导肠黏膜微血管内皮细胞COX-2表达的影响及机制研究

目的探讨姜黄素对LPS诱导的大鼠肠黏膜微血管内皮细胞表达COX-2的影响及机制。方法选用大鼠肠黏膜微血管内皮细胞,随机分组,用p38MAPK抑制剂、PPARγ拮抗剂、姜黄素进行干预。荧光定量RT-PCR检测COX-2 mRNA的表达量,WB法检测其相关蛋白。结果LPS组较正常对照组COX-2增高3.5倍,差异有统计学意义(P0.01)。姜黄素组、p38MAPK选择性抑制剂组、PPARγ拮杭剂组较LPS组,COX-2明显降低。阻断p38MAPK后,COX-2蛋白表达下降。姜黄素可以增加PPARγ、COX-2、p-p38的表达。结论姜黄素可降低COX-2表达水平,p38MAPK、PPARγ通路参与姜黄素调控COX-2的表达。     

巨噬细胞免疫调变信号:Raf-1,MAPK p44,MAPK p42和p38 MAPK的研究

为了了解巨噬细胞免疫调变机理,我们应用LPS和PMA处理小鼠抑制性巨噬细胞,观察到Ras下游信号分子Raf-1,分裂原激活蛋白激酶MAPK p44,MAPK p42和p38 MAPK均被活化,发现forskolin能增强p38 MAPK的活性,进一步提示PKC和PKA途径增强了p38 MAPK的磷酸化效应,为我们了解LPS如何激活p38 MAPK信号通路提供了一个新的机会。     

一氧化碳减轻脂多糖诱导的大鼠小肠损伤与p38 MAPK磷酸化水平的关系

目的：观察低浓度一氧化碳（CO）吸入和腹腔给予对脂多糖（LPS）诱导大鼠小肠损伤的作用及作用过程中丝裂原活化蛋白激酶p38（p38 MAPK）磷酸化水平的变化。方法：6组SD大鼠静脉注入5mg／kg体质量IPS或等容量生理盐水;1h后,对照及LPS注入组吸入室内空气,CO吸入及LPS注入＋CO吸入组吸入体积分数为2．5×10^-4CO．CO腹腔及LPS注入＋CO腹腔组腹腔通入体积分数为2．5×10^-4CO。观察1、3、6h后放血处死,取回盲部上小肠,酶联免疫吸附法测定血小板活化因子（PAV）及细胞间黏附分子-1（ICAM-1）水平;光镜观察组织形态学变化;蛋白印迹法测定p38 MAPK磷酸化水平。结果：LPS注入组PAF、ICAM-1及p38 MAPK磷酸化水平显著高于相应时间点的对照、CO吸入及CO腹腔组（P均〈0．01）;组内各时间点比较,差异无统计学意义。与相应时间点的LPS注入组比较,LPS注入＋CO吸入及LPS注入＋CP腹腔组的PAF和ICAM-1明显降低（P均〈0．05）,但p38 MAPK磷酸化水平进一步增高（P均〈0．05）;此两组间及两组内各时间点比较,差异无统计学意义。结论：低浓度CO吸入和腹腔给予以非时间依赖方式下调LPS诱导的大鼠小肠PAF、ICAM-1表达而起相似的保护作用;p38 MAPK信号转导通路可能参与了这一过程。     

蛋白激酶的生化活性检测方法研究进展

蛋白激酶在真核细胞信号转导中起重要作用,影响了生长、发育、迁移以及凋亡等各个细胞过程。其表达水平或活性异常时,就有可能导致癌症、心血管疾病以及其他各种疾病,因此蛋白激酶是治疗这些疾病很好的分子靶点。迄今为止,美国食品药品监督管理局已经批准了28个蛋白激酶的抑制剂作为上市药物,用于相应的临床治疗。目前存在着各种检测激酶活性的方法,激酶生化检测方法尤为众多,比较经典的有放射性同位素的方法,也有一些非均相非放射性同位素的方法,诸如酶联免疫法、反相高效液相色谱法、核磁共振分析法等等。而各种均相非放射性同位素的检测方法,由于其污染小、操作便捷,逐渐成为激酶抑制剂筛选的首选。本文综述了各种激酶生化检测方法及其发展历史,并介绍了一些新的趋势,如激酶酶谱筛选。     

Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.

We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.     

Molecular aspects of mechanical stress-induced cardiac hypertrophy

To elucidate the signal transduction pathway from external stimuli to nuclear gene expression in mechanical stress-induced cardiac hypertrophy, we examined the time course of activation of protein kinases such as Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90^rsk) in neonatal rat cardiomyocytes. Mechanical stretch rapidly activated Raf-1 and its maximal activation was observed at 1–2 min after stretch. The activity of MAPKK was also increased by stretch, with a peak at 5 min after stretch. In addition, MAPKs and p90^rsk were maximally activated at 8 min and at 10–30 min after stretch, respectively. Next, the relationship between mechanical stress-induced hypertrophy and the cardiac renin-angiotensin system was investigated. When the stretch-conditioned culture medium was transferred to the culture dish of non-stretched cardiac myocytes, the medium activated MAPK activity slightly but significantly, and the activation was completely blocked by the type 1 angiotensin II receptor antagonist, CV-11974. However, activation of Raf-1 and MAPKs provoked by stretching cardiomyocytes was only partially suppressed by pretreatment with CV-11974. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1, MAPKK, MAPKs and p90^rsk, and that angiotensin II, which is secreted from stretched myocytes, activates a part of these protein kinases.Abbreviations MAPK mitogen-activated protein kinase - MAPKK MAP kinase kinase - Raf-1 - Raf- 1 kinase p90^rsk, 90 kDa ribosomal S6 kinase; AngII - angiotensin II - MAPKKK MAP kinase kinase kinase - rMAPK recombinant MAPKK fused to gluthathione S transferase - MMAKK recombinant MAPK fused to maltose binding protein - MBP myelin basic protein - ACE angiotensin-converting enzyme     

Hypotaurocyamine kinase evolved from a gene for arginine kinase

Hypotaurocyamine kinase (HTK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase (AK). HTK is found only in sipunculid worms, and it shows activities for both the substrates hypotaurocyamine and taurocyamine. Determining how HTK evolved in sipunculids is particularly insightful because all sipunculid-allied animals have AK and only some sipunculids have HTK. We determined the cDNA sequence of HTK from the sipunculid worm Siphonosoma cumanense for the first time, cloned it in pMAL plasmid and expressed it in E. coli as a fusion protein with maltose-binding protein. The cDNAderived amino acid sequence of Siphonosoma HTK showed high amino acid identity with molluscan AKs. Nevertheless, the recombinant enzyme of Siphonosoma HTK showed no activity for the substrate arginine, but showed activity for taurocyamine. Comparison of the amino acid sequences of HTK and AK indicated that the amino acid residues necessary for the binding of the substrate arginine in AK have been completely lost in Siphonosoma HTK sequence. The phylogenetic analysis indicated that the HTK amino acid sequence was placed just outside the molluscan AK cluster, which formed a sister group with the arthropod and nematode AKs. These results suggest that Siphonosoma HTK evolved from a gene for molluscan AK. Moreover, to confirm this assertion, we determined by PCR that the gene for Siphonosoma HTK has a 5-exon/4-intron structure, which is homologous with that of the molluscan AK genes. Further, the positions of splice junctions were conserved exactly between the two genes. Thus, we conclude that Siphonosoma HTK has evolved from a primordial gene for molluscan AK.     

Analysis of CaM-kinase signaling in cells

A change in intracellular free calcium is a common signaling mechanism that modulates a wide array of physiological processes in most cells. Responses to increased intracellular Ca²⁺ are often mediated by the ubiquitous protein calmodulin (CaM) that upon binding Ca²⁺ can interact with and alter the functionality of numerous proteins including a family of protein kinases referred to as CaM-kinases (CaMKs). Of particular interest are multifunctional CaMKs, such as CaMKI, CaMKII, CaMKIV and CaMKK, that can phosphorylate multiple downstream targets. This review will outline several protocols we have used to identify which members and/or isoforms of this CaMK family mediate specific cellular responses with a focus on studies in neurons. Many previous studies have relied on a single approach such as pharmacological inhibitors or transfected dominant-negative kinase constructs. Since each of these protocols has its limitations, that will be discussed, we emphasize the necessity to use multiple, independent approaches in mapping out cellular signaling pathways.     

Structural and functional characterization of arabidopsis GSK3-like kinase AtSK12

Plant GSK3-like kinases are key regulators that modulate a broad range of physiological processes such as cell growth, stomatal and flower development, responses for abiotic and biotic stress, and carbohydrate metabolism. Arabidopsis Shaggy/GSK3-like kinases (AtSK) consist of ten members that are classified into four subfamilies (I～IV). Only one of these Arabidopsis GSK3s, BIN2 (also named AtSK21), has been characterized by biochemical and genetic studies. BIN2 acts as a negative regulator in brassinosteroid (BR) signaling that controls cell growth and differentiation. Recent studies suggest that at least seven AtSKs are involved in BR signaling. However, specificities for the substrates and the functional differences of each member of the family remain to be determined. Here we report structural characteristics and distinct function of AtSK12 compared with BIN2. AtSK12 has a longer N-terminal extension, which is absent in BIN2. Transgenic plants overexpressing the AtSK12 mutant carrying deletion of Nterminal region display more severe dwarf phenotypes than those of the wild-type AtSK12. Microscopic analysis reveals that N-terminal-deleted AtSK12 accumulates in the nucleus. This implies that structural difference in the Nterminal region of AtSK members contributes to their subcellular localization. In contrast to BIN2, overexpression of AtSK12 does not cause a stomatal cluster. Furthermore, we show that YODA MAPKKK, which controls stomatal development, interacts with BIN2 but not with AtSK12. Our results suggest that AtSK12 mediates BR-regulated cell growth but not stomatal development while BIN2 regulates both processes. Our study provides evidence that different GSK3 members can have overlapping but non-identical functions.     

The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase

The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km^Tc and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.     

Induced fit in guanidino kinases--comparison of substrate-free and transition state analog structures of arginine kinase

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 A with a final R of 22.3% (R(free) = 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.     

Activation of Arabidopsis MAPK kinase kinase (AtMEKK1) and induction of AtMEKK1–AtMEK1 pathway by wounding

We have constructed a series of deletion mutants of Arabidopsis MAPK kinase kinase (AtMEKK1) and obtained a constitutively active mutant, AtMEKK1Δ166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Δ166 predominantly phosphorylates AtMEK1, an Arabidopsis MAPKK, but not its double mutant (AtMEK1T218A/S224E), suggesting that Thr-218 and Ser-224 are the phosphorylation sites. In wounded seedlings, AtMEKK1 was activated and phosphorylated its downstream AtMEK1. Furthermore, analysis using anti-AtMEKK1 and anti-AtMEK1 antibodies revealed that the interaction between the two proteins was signal dependent. These results suggest the presence of AtMEKK1–AtMEK1 pathway induced by wounding.