苹果和葡萄果实蛋白激酶特性分析

以组蛋白Ⅲ－Ｓ作苹果和葡萄果肉蛋白激酶制剂底物时,反应体系中加ＥＧＴＡ可抑制蛋白激激酶活性,而加Ｃａ＾２＋可激活蛋白激酶的活性,而且,葡萄果实微粒体蛋白激酶钙的蛋白激酶存在。而且,葡萄果实微粒体蛋白激酶呈热稳定性,苹果果实微粒体蛋白激酶对热敏感。以髓鞘碱性蛋白（ＭＢＰ）作底物,在苹果和葡萄果实微粒体中都检测出很高的蛋白酶活性,并且不依赖于钙,说明苹果和葡萄果实中可能有分裂原激活的蛋白激酶（ＭＡＰ激     

苹果和葡萄果实发育过程中蛋白激酶特性的比较研究

在苹果 (MalusdomesticaL .Borkh .cv .RedStarking)和葡萄 (VitisviniferaL .×V .lubruscaL .cv .Kyoho)的不同发育阶段均检测到蛋白激酶活性 ,它们可被Ca2 强烈激活 ,表明依赖钙的蛋白激酶是苹果和葡萄果实中一类非常重要的蛋白激酶。钙调素 (CaM)对苹果和葡萄果实蛋白激酶没有激活作用 ,同时CaM拮抗剂calmidazolium、W7及三氟乙酸 (TFA)均可不同程度地抑制其活性 ,表明依赖钙的蛋白激酶中可能有含类似钙调素结构域的蛋白激酶(CDPK)的存在。在苹果和葡萄果实发育中 ,依赖钙的蛋白激酶无论在其活性变化还是特性上都存在着显著差异 :苹果果实发育的不同阶段依赖钙的蛋白激酶活性无明显变化 ,而葡萄果实在发育的第Ⅱ期蛋白激酶活性大大高于第Ⅰ、Ⅲ期 ;苹果果实依赖钙的蛋白激酶可被Mn2 强烈激活并对热敏感 ,而葡萄果实依赖钙的蛋白激酶却不受Mn2 及热处理的影响 ;此外 ,葡萄果实蛋白激酶活性不受磷脂酰丝氨酸 (PS)的影响 ,而苹果果实蛋白激酶可被PS激活 ,表明苹果果实中还可能有钙和磷脂激活的蛋白激酶即PKC的存在。苹果和葡萄果实发育中蛋白激酶种类、特性及其活性变化的不同 ,暗示了这两种果实发育调控的差异 ,也意味着苹果和葡萄果实发育调控可能与蛋白激酶密切相关。     

盐胁迫对玉米根尖质膜上受钙激活的蛋白激酶的影响

以02mal／L的NaCl对玉米根尖进行盐胁迫,导致质膜上一种受钙激活的蛋白激酶（该激酶表现为钙依赖蛋白激酶的特性）的活性迅速增加。盐胁迫至15min时激酶的活性达到最大,较对照高30％,以后逐渐降低,盐胁迫至切50min时激酶活性仍略高于对照。10％PEG6000处理玉米很尖也可诱导质膜上受钙激活的蛋白激酶活性增加。用外源ABA处理玉米根尖,未导致上述蛋白激酶活性的变化。放射自显影显示质膜上33kD和58kD两种蛋白可能是质膜受钙激活的蛋白激酶的内源底物。     

玉米根尖质膜的受钙激活蛋白激酶的特性

以生长3－4d的玉米（ZeamaysL．）根尖为材料,用两相法得到高纯度的质膜,鉴定了质膜上存在一受钙激活的蛋白激酶。该类激酶主要位于质膜内侧;钙的半激活浓度为50μmol／L;外源钙调素对激酶没有明显的活化作用;钙调素拮抗剂三氟拉嗪（TFP）可明显抑制激酶活性,半抑制浓度为75μmol／L;该类激酶对外源底物组蛋白ⅢS型有较高的特异性。结果显示此激酶属于钙依赖钙调素不依赖蛋白激酶。质膜上33kD和58kD两种蛋白可能是这种钙依赖蛋白激酶的内源底物。     

大豆叶片质膜蛋白激酶的自身磷酸化反应

利用Ferrell和Martin（1991）设计的测定印迹在PVDF膜上的蛋白激酶活性方法研究大豆叶片质膜蛋白激酶自身磷酸化反应活性,结果表明：与Mg－ATP相比,Mn－ATP是更有效的57KD蛋白激酶自身磷酸化反应底物;钙离子可以促进该激酶的自身磷酸化反应活性,而且EGTA可以显著降低它在SDS电泳中的迁移率,说明57KD蛋白激酶为依赖于钙的蛋白激酶;预磷酸化反应实验证明57KD蛋白激酶具有多个自身磷酸化反应位点,其分子的自身磷酸化状态可调性暗示这一激酶可能具有重要的生理功能。     

大豆叶片质膜蛋白激酶的自身磷酸化反应

利用Ｆｅｒｒｅｌｌ和Ｍａｒｔｉｎ设计的测定印迹在ＰＶＤＦ膜上的蛋白激酶活性方法研究大豆叶片质膜蛋白激酶自身磷酸化反应活性,结果表明：与Ｍｇ－ＡＴＰ相比,Ｍｎ－ＡＴＰ是更有效的５７ｋＤ蛋白激酶自身磷酸化反应底物;钙离子可以促进该激酶的自身磷酸化反应活性,而且ＥＧＴＡ可以显著降低它在ＳＤＳ电泳中的迁移率,说明５７ｋＤ蛋白激酶为依赖于钙的蛋白激酶;     

高温胁迫下葡萄叶片蛋白激酶的诱导形成与活性变化

以"京秀"葡萄(Vitis vinifera L.cv.Jingxiu)幼苗为试材,研究了高温胁迫激活的蛋白激酶的类型和活性.结果表明,高温胁迫10～60min明显地激活了一个分子量约为52 kD的蛋白激酶,该蛋白激酶能将凝胶中所嵌入的髓鞘碱性蛋白(MBP)磷酸化,在放射自显影中表现出很高的放射活性,而对凝胶中的组蛋白-Ⅲ(histone-Ⅲ)则没有这样的作用.在溶液反应体系中该蛋白激酶对MBP也表现出很高的磷酸化活性,而对histone-Ⅲ却无作用.Ca2 对其活性变化无显著影响.酪氨酸特异性蛋白磷酸酶(YOP)对该激酶的活性有显著的钝化作用.结果表明该52 kD蛋白激酶是MAPK家族中的一种.     

蛋白磷酸酶-1对凋亡酶-3水解的PAK2活性的负调控机制

p21激活的蛋白激酶2(PAK2)可以通过与小G蛋白Cdc42和Rac结合而激活, 也可以通过凋亡酶-3(caspase-3)的水解作用而激活. PAK2的这2种激活方式都需要其402位苏氨酸残基(Thr-402)发生自磷酸化. 对于PAK2激活的研究已经有近10年的时间, 但PAK2活性的负调控机制仍然不清楚. 本文采用酶活性不可逆改变动力学的方法研究了蛋白磷酸酶1催化亚基(PP1a)对凋亡酶-3(caspase-3)水解的PAK2活性的调控机制. 根据在PP1a存在下PAK2的底物反应动力学方程, 我们确定了游离酶和酶-底物复合物的微观失活动力学常数. 结果表明: (ⅰ) PP1a可以直接使PAK2活化环上的Thr-402去磷酸化, 从而下调PAK2的激酶活力; (ⅱ)外源蛋白或多肽底物结合在PAK2活性位点上可以降低PAK2自激活和失活的速度. 本文所建立的方法不仅可以用于研究酶活性调节的修饰反应动力学, 还可以研究底物对修饰反应的影响, 为研究蛋白激酶和磷酸酶的作用机理奠定了理论基础.     

DNA依赖蛋白激酶研究进展

DNA依赖蛋白激酶由Ku异二聚体和DNA-PKcs组成,结合Ku蛋白后,DNA-PK激酶活性激活,DNA依赖蛋白激酶具有多功能性,参与DNA修复、基因重组以及复制、转录等多种细胞学过程.     

绿藻Dunaliella salina钙依赖蛋白激酶的特征(英文)

通过凝胶过滤从绿藻Dunaliella salina中分离出一种新类型的钙依赖但非钙调素、磷脂依赖的蛋白激酶,用凝胶过滤法测得此酶的天然分子量为52kD。该酶的活性既依赖于Ca~(2 )也需要Mg~(2 ),最适浓度为4mmol/L。NaCl和KCl对酶活性具抑制作用。蛋白酶抑制剂K-252a和staurosporine可抑制该酶活性,但半抑制浓度 (IC_(50))远高于对蛋白激酶C(PKC)。当PKC专一性抑制剂sphingosine浓度高达800μmol/L时,对该酶只表现微弱的抑制效应。碱性的组蛋白H1为所测定的蛋白质底物中最适的底物,而酸性的酪蛋白不被磷酸化。磷酸化氨基酸残基分析表明,该酶属丝氨酸/苏氨酸型蛋白激酶。     

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.     

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.     

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。     

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.     

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     

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.     

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.     

The Mck1 GSK-3 kinase inhibits the activity of Clb2-Cdk1 post-nuclear division

The glycogen synthase kinase-3 homolog, Mck1, has been implicated in many cellular functions, from sporulation to calcium stress response in budding yeast. Here, we report a novel function for Mck1 in the inhibition of Clb2-Cdk1 activity post nuclear division. Clb2-Cdk1, the major mitotic cyclin-Cdk complex in yeast, accumulates before anaphase and must be inhibited in telophase for cells to exit mitosis and enter into the next cell cycle. We show that the mck1Δ mutant is highly sensitive to increased Clb2-Cdk1 activity caused either by overexpression of Clb2 or the Cdk1-activating phosphatase Mih1. Deletion of the Cdk1 inhibitory kinase, SWE1, in combination with a mck1Δ mutant results in a synthetic growth defect, suggesting that Mck1 and Swe1 function in parallel pathways to inhibit Clb2-Cdk1. We find that mck1Δ strains have a delay in mitotic exit as well as elevated levels of Clb2-Cdk1 activity post-nuclear division. Using a co-immunoprecipitation assay, we identify a physical interaction between Mck1 and both Clb2 and Mih1. Finally, we demonstrate that phosphorylation of purified Clb2 by Cdk1 is inhibited by catalytically active Mck1 but not catalytically inactive Mck1 in vitro. We propose that Mck1 inhibits the activity of Clb2-Cdk1 via interaction with Clb2. The mammalian glycogen synthase kinase-3 homolog has been implicated in cyclin inhibition, suggesting a conserved cell cycle function for both yeast and mammalian glycogen synthase kinases.     

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.