Latent Periodicity of Serine/Threonine and Tyrosine Protein Kinases and Other Protein Families

A method of noise decomposition has been developed. This method allows for the identification of a latent periodicity with symbol insertions and deletions that is specific for all or most amino acid sequences belonging to the same protein family or protein domain. The latent periodicity has been identified in catalytic domains of 85% of serine/threonine and tyrosine protein kinases. Similar results have been obtained for 22 other protein families. The possible role of latent periodicity in protein families is discussed.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 420–436.Original Russian Text Copyright © 2005 by Laskin, Kudryashov, Skryabin, Korotkov.     

Genome-Wide Analysis and Experimentation of Plant Serine/ Threonine/Tyrosine-Specific Protein Kinases

Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.     

丝裂原活化蛋白激酶激活蛋白激酶(MK)研究进展

丝裂原活化蛋白激酶(MAPK)信号通路介导多种重要的细胞生理反应.对下游蛋白激酶的磷酸化是MAPK家族成员发挥生理作用的重要方式.在MAPK的下游存在3个结构上相关的MAPK激活蛋白激酶(MAPKAPKorMK),即MK2,MK3和MK5.在被MAPK激活后,MK可将信号传递至细胞内不同靶标,从而在转录和翻译水平调节基因表达,调控细胞骨架和细胞周期,介导细胞迁移和胚胎发育.最近,在基因敲除研究的基础上,不同MK亚族成员之间的功能区分已经逐渐明晰,使我们对于MK的认识有了长足的进步.     

Protein Tyrosine Kinases in Human Brain and Gliomas

Tyrosine kinase activity was determined in neonatal and adult human brain, oligodendrogliomas, and astrocytomas. The astrocytomas were divided into low- (grade I and grade II) and high-grade (grade III and grade IV) tumors. We measured the tyrosine kinase activity in the cytosolic and membrane fraction using poly(glutamic acid:tyrosine, 4:1) as an artificial substrate. The cytosolic activity in oligodendrogliomas (n = 7), low-grade astrocytomas (n = 7), and neonatal brain (n = 1) was increased, on average, two- to fourfold compared with that in normal adult brain (n = 14). The cytosolic activities of high-grade astrocytomas (n = 11) were in approximately the same range as found in normal adult brain. The absence of an increase in cytosolic activity in high-grade astrocytomas compared with adult brain is likely due to the occurrence of necrosis in these tumors. In contrast to the cytosolic activity, no differences were found in the membrane-bound activity. By fast protein liquid chromatography, at least three forms of cytosolic protein tyrosine kinase could be separated, which eluted at 0, 115, and 210 mM NaCl. In most cases the highest amount of activity eluted at 210 mM NaCl. However, in oligodendrogliomas, high-grade astrocytomas, and neonatal brain, more activity eluted at 115 mM NaCl than in normal adult brain (p = 0.043). Nevertheless, protein tyrosine kinases from all three peaks contributed to the elevated levels of total cytosolic activity of oligodendrogliomas and low-grade astrocytomas.     

Variable Interactions between Sucrose Non-fermented 1-Related Protein Kinases and Regulatory Proteins in Higher Plants

WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.     

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。     

PTP及其在植物MAPK途径中的作用

蛋白酪氨酸磷酸酶（protein tyrosine phosphatases,PTPs）是一个结构多样的磷酸酶家族,含有高度保守的催化结构域。在植物体内,PTP主要的靶蛋白是促细胞分裂剂激活性蛋白激酶（mitogen-activated protein kinase,MAPK）。MAPK级联途径参与有机体的发育、细胞增殖、激素调节以及逆境胁迫的信号转导,PTP在MAPK级联途径中主要起负调控作用。本文就PTP的结构和功能、MAPK在植物中的作用及PTP在MAPK级联途径中的功能进行综述,并着重介绍PTP在拟南芥中的研究进展。     

Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases

The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.     

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

大豆叶片衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化

以大豆幼苗初生叶为材料研究了衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化。结果发现质膜上一个57kD的蛋白激酶分子上有多个自磷酸化位点,而且自磷酸化反应能提高该酶催化组蛋白H1磷酸化的激酶活力。进一步的研究表明诱导衰老处理造成的57kD蛋白激酶自磷酸化状态的变化,可能对调节它在衰老过程中催化活性的变化起重要作用;而外源6-BA预处理则能够维持57kD蛋白激酶体内高自磷酸化状态,保持该激酶在衰老过程中的催化活力。对衰老和6-BA处理过程中质膜上39和47kD蛋白激酶自磷酸化状态变化的研究表明,这两种激酶可能参与大豆叶片对6-BA刺激信号的传导和/或应答反应过程。     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

Serine/Threonine Protein Kinases from Bacteria,Archaea and Eukarya Share a Common Evolutionary Origin Deeply Rooted in the Tree of Life

The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al. in 1988 (Science, 241, 42–52). It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reasons, the term “eukaryotic-type kinases” propagated in the literature to describe bacterial members of this protein family. Here, we argue that this term should be abandoned as a misnomer, and we provide several lines of evidence to support this claim. Our comprehensive phylostratigraphic analysis suggests that Hanks-type kinases present in eukarya, bacteria and archaea all share a common evolutionary origin in the lineage leading to the last universal common ancestor (LUCA). We found no evidence to suggest substantial horizontal transfer of genes encoding Hanks-type kinases from eukarya to bacteria. Moreover, our systematic structural comparison suggests that bacterial Hanks-type kinases resemble their eukaryal counterparts very closely, while their structures appear to be dissimilar from other kinase families of bacterial origin. This indicates that a convergent evolution scenario, by which bacterial kinases could have evolved a kinase domain similar to that of eukaryal Hanks-type kinases, is not very likely. Overall, our results strongly support a monophyletic origin of all Hanks-type kinases, and we therefore propose that this term should be adopted as a universal name for this protein family.     

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.     

酪氨酸蛋白激酶和蛋白激酶C对N-乙酰氨基葡萄糖转移酶V的双重调控

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮（ｇｅｎｉｓｔｅｉｎ）在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的基础活力,并完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的增高作用;ＴＰＫ的特异性抑制剂Ｔｙｒｐｈｏｓｔｉｎ－２５和ＰＫＣ的特异性抑制剂Ｄ－鞘氨醇分别应用时,各自只能部分地取消ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导。但当Ｔｙｒｐｈｏｓｔｉｎ－２５和Ｄ－鞘氨醇同时加入培养基中则可完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制ＧｎＴ－Ｖ的基础活力,也可完全消除ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的激活。以上结果提示ＥＧＦ或ＰＭＡ通过蛋白激酶调节ＧｎＴ－Ｖ的酶蛋白合成,并且ＧｎＴ－Ｖ受到膜性ＴＰＫ和ＰＫＣ的双重调节,其中ｍ－ＴＰＫ较ｍ－ＰＫＣ更为重要。     

Role of Extracellular Signal-Regulated Protein Kinases 1 and 2 in Oligodendroglial Process Extension

Abstract: The relationship between extracellular signal-regulated protein kinase (ERK) activation and process extension in cultured bovine oligodendrocytes (OLGs) was investigated. Process extension was induced through the exposure of cultured OLGs to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), for various intervals. During the isolation of these OLGs from bovine brain, the original processes were lost. Therefore, any reinitiation of process extension via PMA stimulation was easily discernible through morphological monitoring. It was found that exposure of OLGs to PMA for 10 min was enough to induce OLG process extension 24–72 h later. Furthermore, this extension was still evident at least 1 week after the initial PMA stimulation, indicating that OLGs do not need continuous PKC activation to sustain process extension. Control and PMA-stimulated OLGs were also subjected to immunocytochemistry using an anti-ERK antibody selective for the mitogen-activated protein kinases p42 Erk2 (ERK2) and p44 Erk1 (ERK1) isoforms. ERK immunoreactivity in the nucleus was evident after PMA stimulation of OLGs but not in control OLGs. In parallel experiments, the control and PMA-stimulated OLGs were purified by Mono Q fractionation and subjected to ERK phosphotransferase assays using [γ-³²P]ATP and either myelin basic protein (MBP) or a synthetic peptide substrate based on the Thr⁹⁷ phosphorylation site in MBP. These assays indicated that in PMA-treated OLGs, ERK activation was at least 12-fold higher than in control OLGs. Anti-ERK and anti-phosphotyrosine western blots of the assay fractions verified an enhanced phosphorylation of ERK1 and ERK2 in PMA-treated fractions relative to control fractions. When OLGs were pretreated for 15 min with the ERK kinase (MEK) inhibitor PD 098059 before PMA stimulation, they exhibited a 67% decrease in ERK activation as compared with cells treated with PMA alone. Furthermore, these MEK inhibitor-pretreated cells were still viable but showed no process extensions up to 1 week later. Therefore, we propose that a threshold level of ERK activity is required for the initiation of OLG process extension.     

植物抗逆相关蛋白激酶的结构与功能

植物在遭受外界逆境胁迫时,体内的信号传导系统能够感知、传递逆境胁迫信号,并引起各种生理生化反应以适应环境。植物蛋白激酶在信号感知、传导以及基因的表达调控中起重要的作用。蛋白激酶在信号传导过程的功能是磷酸化修饰目的蛋白,而磷酸化的实现需要蛋白质之间相互作用。本文从植物蛋白激酶的结构、分类、与激素信号传导之间的关系等方面进行了系统的阐述,对蛋白激酶介导的植物抗性与发育的最新研究进展进行了系统的总结,为解析蛋白激酶在植物生长发育中的抗逆机理提供依据。     

植物逆境胁迫相关蛋白激酶的研究进展

干旱、高盐、高温和低温等非生物胁迫及各种病虫害等生物胁迫严重影响植物的生长发育和作物产量.蛋白激酶主要通过激活不同的磷酸化途径介导外界环境信号的感知和传递,调控下游抗逆基因的转录表达,启动相应的生理生化等适应性反应来降低或消除危害.该文对近年来国内外有关与非生物胁迫和生物胁迫信号传导相关的受体蛋白激酶、促分裂原活化蛋白激酶、钙依赖而钙调素不依赖的蛋白激酶、蔗糖不发酵相关蛋白激酶和其它胁迫相关的植物蛋白激酶的研究进展进行综述,探索蛋白激酶介导的不同磷酸化途径应对逆境胁迫的信号传递网络,为进一步了解植物逆境分子应答机制提供依据.     

植物类受体蛋白激酶的研究进展

植物类受体蛋白激酶(receptor-like protein kinase,RLKs)通过胞外结构域识别病原信号分子,发生磷酸化、去磷酸化反应而开启或关闭下游靶蛋白,调节植物固有免疫反应,诱导抗病防御反应.目前对植物类受体蛋白激酶的功能、信号传导和配体识别等方面的研究已成为该领域的重点.本文对近年来国内外有关植物类受体蛋白激酶的结构、功能及其在植物抗病防御反应中的作用研究进行综述,为今后进一步深入研究植物类受体蛋白激酶的生理生化功能及应用提供参考.