植物凝集素类受体激酶的研究进展

植物凝集素类受体激酶是定位在细胞膜上的蛋白质,其结构从胞外至胞内依次是氨基端信号肽、配体结合域、单通道跨膜域和胞内丝氨酸/苏氨酸激酶域.目前对植物凝集素类受体激酶的功能、信号转导和配体识别等方面的研究已成为该领域的重点.对近年来国内外有关植物凝集素类受体激酶的结构、分类及其在植物抗病防御和抗逆反应中的作用研究进行综述,为今后进一步深入研究植物凝集素类受体激酶的生理生化功能及应用提供参考.     

利用酵母双杂交系统筛选水稻类受体激酶OsWAK50胞内域相互作用蛋白

细胞壁连接的类受体激酶(wall-associated kinase,WAK)是植物细胞中一类特有的类受体激酶基因亚家族,因其胞外域与细胞壁紧密相连而得名．水稻中共有125个OsWAK基因,OsWAK50编码的蛋白质具有胞外域、跨膜域和激酶域,呈现典型的WAK样受体激酶特征．首先通过对OsWAK50-GFP融合蛋白的观察发现OsWAK50定位于细胞膜并且与细胞壁偶联．进而通过酵母双杂交系统筛选到了20个可能与OsWAK50胞内域相互作用的候选蛋白,并通过一对一酵母转化验证了OsSK4、OsSWIB和OsSWI3C全长均可与OsWAK50胞内域相互作用．进一步分析显示,OsSWIB能够直接与OsWAK50激酶域互作,而OsSK4和OsSWI3C与OsWAK50胞内域的互作是依赖于OsWAK50 C端的．研究还表明,OsSK4和OsSWIB亦能与OsWAK50同源基因OsWAK53a结合,而OsSWI3C则不能与OsWAK53a结合．双分子荧光互补实验证明,OsSK4与OsWAK50和OsWAK53a能够在植物体内发生互作．以上结果为阐明OsWAK50发挥功能的分子机制提供了重要线索．     

XA21和PI-D2激酶蛋白质在酿酒酵母中的表达及其自我磷酸化研究

白叶枯病和稻瘟病是最主要的水稻病害。Xa21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质。在前期研究中,曾系统地研究了细菌中表达XA21激酶蛋白质的生化活性。在此实验中利用真核表达系统酿酒酵母对Xa21和Pi-d2编码的蛋白激酶进行了表达、纯化及自我磷酸化活性分析,为进一步的生化分析、蛋白质-蛋白质相互作用研究、底物筛选等奠定了基础。     

类受体激酶调控根生长发育的研究进展

类受体激酶是一类具有激酶活性的单次跨膜受体,通过接收和传递胞外信号调控细胞的生理反应,参与植物生长发育过程。植物根在生长发育过程中受到大量的外部刺激和内源性发育信号的影响,植物必须通过整合这些信号并转化为细胞反应,才能适应不断变化的环境条件;植物类受体激酶作为细胞膜上的信息监测者,通过与外源和内源信号的通讯调控根的生长发育。该文对近年来国内外有关类受体激酶的结构、分类及其作用机制,特别是植物类受体激酶在根发育信号转导途径中的功能和作用等方面的研究进展进行综述,为进一步揭示植物类受体激酶在根生长发育中的功能及其作用机制提供参考。     

植物富含半胱氨酸的类受体激酶的研究进展

类受体激酶(receptor-like kinase,RLK)是植物体内普遍存在的一类蛋白激酶,是许多信号识别传递途径中的关键组分,作为识别信号的质膜受体,能够感受外界刺激,通过磷酸化作用参与胞内信号传递。富含半胱氨酸的类受体激酶(cysteine-rich receptor-like kinase,CRK)又被称为DUF26(domain of unknown function 26)类受体激酶,是RLK中的一大类。近几年,越来越多的研究发现CRK参与了植物的抗病防御反应。综述了植物CRK蛋白的结构特征,总结了目前已发现的CRK在生物胁迫、非生物胁迫及生长发育中的功能,并对CRK未来的研究进行了展望,旨为后续阐明CRK的功能和分子机制提供参考。     

类受体蛋白激酶在植物中的研究进展

植物体在接收外界信号分子时,这些细胞外信号被细胞膜上受体特异性相结合,通过体内一系列信号转导途径将生物信号进行放大或传递,引起相应的生物效应,从而完成植物体需要进行的生命活动。类受体蛋白激酶(receptor-like protein kinases,RLKs)定位在细胞质膜上,由胞内区、跨膜区和胞外区3部分构成。RLKs的工作机理主要是通过胞外信号分子与其胞外区结构域特异结合,结合后激活胞内激酶域而完成跨膜信号的转导。在植物体内能够参与信号转导、抗逆反应和病原反应等途径,对植物体具有重要意义。本综述将对植物RLKs的结构、分类及生理功能方面进行分析,为深入研究RLKs提供理论基础。     

五个水稻类受体激酶的抗体制备及检测

类受体激酶在植物发育、自交不亲和、雄性不育、抗逆和抗病等生命过程中起着重要的调控作用。为了对水稻类受体激酶的功能及生物学特性进行深入研究,本实验克隆表达了水稻中5个类受体激酶抗原表位片段,以纯化的蛋白质为抗原免疫新西兰兔,获得了特异性较高的多克隆抗体。Western blotting检测结果表明,5个类受体激酶均在叶片中表达。     

表皮生长因子受体酪氨酸激酶的克隆表达和酶抑制剂筛选模型的构建

表皮生长因子受体（epidermal growth factor receptor,EGFR）属受体酪氨酸激酶（tyrosine kinase,TK）家族,其胞内的酪氨酸激酶在细胞信号转导通路中具有十分重要的作用。许多肿瘤的发生、发展都与EGFR胞内酪氨酸激酶的异常表达密切相关。因此,EGFR胞内酪氨酸激酶的抑制剂有可能成为治疗肿瘤的有效药物。从人脐静脉内皮细胞（HUVEC）提取总RNA,采用RT-PCR获得EGFR酪氨酸激酶催化域的编码基因。将其克隆至载体pET-30a,在E．coliBL21（DE3）中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化。通过对酶的活性的测定,证明重组EGFR酪氨酸激酶蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性。以该重组激酶为靶位构建了酶抑制剂筛选模型,拟对微生物代谢产物进行筛选。     

大豆类受体蛋白激酶基因GmRLCK的克隆及初步分析

植物类受体蛋白激酶(receptor-likeproteinkinase,RLK)在高等植物生长发育和环境刺激的信号传导中起着重要的作用。本文报告了一个新的大豆类受体蛋白激酶基因的全长cDNA克隆及对其基因结构和功能的初步分析。研究表明该基因序列编码的蛋白包含一个跨膜域、一个具有丝氨酸/苏氨酸激酶活性的胞内域和一个缺少N-末端信号肽的胞外域。采用生物信息学方法分析表明,该基因与一些拟南芥菜类受体蛋白激酶基因具有很高的相似性,这些激酶N-末端都缺少信号序列,属于植物胞质类受体激酶(receptor-likecytoplasmickinase,RLCK)亚家族。因此命名该大豆基因为GmRLCK(GenBankAccessionNo.AY687390)。对GmRLCK激酶域中磷酸化可能性较高的位点进行了预测。RT-PCR的结果表明,GmRLCK在大豆子叶、根、花以及豆荚中都有较高的表达,而在胚根、茎和成熟叶片中的表达相对较弱。进化分析表明GmRLCK与一些衰老相关的植物类受体蛋白激酶具有较近的亲缘关系。     

植物类受体胞质激酶的结构和功能

本文介绍植物类受体胞质激酶的结构及其在植物的抗病、抗逆、生长发育、自交不亲和、油菜素内酯信号转导等方面的功能。     

QIKS – Quantitative identification of kinase substrates

Signaling networks regulate cellular responses to external stimuli through post‐translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large‐scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel‐screening platform developed for the proteome‐wide substrate‐analysis of specific kinases. Here, we aimed to identify substrates of mitogen‐activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen‐activated protein kinase cascade. An ATP analog‐sensitive mutant of Mek1 (Mek1‐as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC‐MS/MS of IMAC‐enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.     

Identification of tyrosine 67 in bovine brain myelin basic protein as a specific phosphorylation site for thymus p56lck.

The protein-tyrosine kinase p56lck exhibits a restricted substrate specificity in vitro but can efficiently phosphorylate bovine myelin basic protein (MBP). Results obtained from both peptide mapping and fast atom bombardment mass spectrometry indicate that tyrosine 67 in the sequence -Thr-Thr-His-Tyr67-Gly-Ser-Leu-Pro-Gln-Lys- in bovine MBP is the specific phosphorylation site. p56lck does not phosphorylate the acidic, cytoplasmic domain of erythrocyte band 3. In contrast, p40, another protein-tyrosine kinase purified from bovine thymus that readily phosphorylates band 3, does not phosphorylate MBP. Therefore, MBP and band 3 may prove to be useful substrates for distinguishing between various tyrosine kinases on the basis of substrate specificity. In addition, identification of the recognition sequence in MBP for p56lck may contribute to an understanding of the structural features of physiological substrates for this kinase.     

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.     

A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain

This study describes a method for the identification of the substrates of specific serine kinases. An antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method was applied to the identification of substrates for the protein kinase Akt, which specifically phosphorylates the RXRXXS/T motif. 3T3-L1 adipocytes were treated with insulin to activate Akt, and the putative Akt substrate proteins were isolated by immunoprecipitation with an antibody against the phospho form of this motif. This led to the identification of a novel 160-kDa substrate for Akt. The 160-kDa substrate for Akt, which was designated AS160, has a Rab GAP domain. Recombinant AS160 was shown to be a substrate for Akt, and two sites of phosphorylation, both in RXRXXS/T motifs, were identified by mass spectrometry and mutation. Insulin treatment of adipocytes caused AS160 to redistribute from the low density microsomes to the cytosol.     

A MAP kinase docking site is required for phosphorylation and activation of p90(rsk)/MAPKAP kinase-1

Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates [1] [2] [3] [4]. The MAP kinase-activated protein (MAPKAP) kinase p90 rsk contains two kinase domains [5]: the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes [6] [7] [8], where inactive p90(rsk) is bound to the inactive form of the Erk2- like MAP kinase p42(mpk1). Here, we identify a new MAP kinase docking site located at the carboxyl terminus of p90(rsk). This docking site was required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and was also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site was conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases.     

Substrate specificity of protein kinases and computational prediction of substrates

To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of "peptide specificity" of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information.     

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

Protein phosphorylation in cultured endothelial cells

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.     

Kinase-interacting substrate screening is a novel method to identify kinase substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.     

Profiling substrate phosphorylation at the phosphopeptide level

The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i). the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii). the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii). the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the gamma-isotype of p21-activated protein kinase (gamma-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of gamma-PAK.