植物蛋白质磷酸化的研究技术

本文介绍植物蛋白质磷酸化研究的技术及其应用情况,并对这些技术的应用前景作了展望。     

综述与专论：蛋白质组氨酸磷酸化修饰的调节及功能作用

组氨酸磷酸化（pHis）在原核和真核生物的生命活动中发挥重要调控作用,并与包括恶性肿瘤在内的多种病理过程相关。pHis修饰中磷酰胺键在高温和低pH下容易断裂,其高度不稳定性导致对pHis修饰的鉴定和研究进展缓慢。近年来,磷酸化蛋白质组学新技术的发展以及pHis特异性抗体的出现,推动了pHis修饰蛋白底物的鉴定和功能研究。首次在哺乳动物细胞中鉴定到超过700个pHis修饰蛋白,并陆续发现黏着斑激酶（FAK）和磷酸甘油酸变位酶1（PGAM1）等蛋白质的pHis修饰能促进肿瘤发展。本文主要探讨组氨酸激酶和组氨酸磷酸酶在调控特定蛋白质pHis修饰中的关键机制及其功能,以期为pHis修饰蛋白的生物学功能研究奠定基础。     

用蛋白质组学方法解析磷酸化蛋白质

蛋白质磷酸化和去磷酸化这一可逆过程参与了高等真核生物细胞信号转导、细胞分化和细胞生长等重要过程,并与许多疾病、肿瘤的发生密切相关。蛋白质组学技术的不断发展和完善,可以更好、更多地识别和鉴定磷酸化蛋白质,为解析磷酸化蛋白质提供了可能。章综述了用于分离和鉴定磷酸化蛋白质的蛋白质组学方法。     

不同品系小菜蛾成虫脑突触体蛋白质磷酸化的研究

对小菜蛾Plutella xylostella(L.)敏感品系、抗溴氰菊酯品系、抗杀虫双品系和抗杀螟丹品系的成虫脑突触体蛋白质磷酸化进行了研究比较。结果表明：蛋白质磷酸化在各个品系中的表现是不一样的。cAMP和钙加钙调蛋白对不同品系小菜蛾脑蛋白质磷酸化都有不同程度的刺激作用;3种杀虫剂均对各品系小菜蛾的磷酸化反应有影响,杀虫双、杀螟丹表现为抑制,溴氰菊酯表现为加强。这种影响在敏感品系中表现得比抗性品系中要强烈。     

杨树蛋白质磷酸化位点预测

以小黑杨磷酸化蛋白质组为研究对象,用人工神经网络表达丝氨酸、苏氨酸等残基位点的磷酸化与氨基酸序列的结构特征之间的非线性关系,建立了BP人工神经网络模型,并用磷酸化数据对所建模型进行训练和分析,得适宜的结构为21×16∶8∶4,拟合准确度为90%,Acc、Sn、Sp、MCC分别为78%、89%、67%、0.57,对比分析结果表明,所建模型具有较强的预测能力。     

蛋白质组中蛋白质磷酸化研究进展

随着后基因组时代的到来 ,对生命体器官、组织或细胞的全部蛋白质的表达、修饰及相互作用的研究已成为蛋白质组学的重要任务。蛋白质磷酸化是细胞内信号转导和酶调控最常见的机制之一 ,人类基因组约 2 %的基因编码 5 0 0种激酶和 10 0种磷酸酶。蛋白质磷酸化和去磷酸化作为原核和真核细胞表达调控的关键环节 ,了解其对功能的影响可以深入理解生命系统在分子水平的调控状况。目前蛋白质组磷酸化研究仍是功能基因组面临的重大课题 ,本文对此作一综述     

蛋白质组氨酸磷酸化研究进展

摘要：蛋白质磷酸化是一种可逆的翻译后修饰,这种翻译后修饰可以改变蛋白质的构象,进而使蛋白质活化或者失活。组氨酸磷酸化在细胞信号传导过程中发挥着重要作用,且组氨酸磷酸化与人类某些疾病密切相关,然而,由于组氨酸磷酸化含有P-N键,具备不稳定性,有关于组氨酸磷酸化的报道远远少于其它磷酸化的报道。本综述系统的总结了组氨酸磷酸化在生物学过程中的作用,以及近些年取得的重要研究进展,以期对深入研究组氨酸磷酸化提供理论参考。     

受体及离子通道蛋白磷酸化对跨膜信号传递的调节

跨膜信号转导是细胞信息传递的起始环节,受体和离子通道在此环节上起重要作用,受体和通道蛋白易受多种因素的调节。蛋白南磷酸化是受体及离子通道调节的关键步骤,不仅使受体及离子通道的功能发生改变,而且地影响到其在细胞的分布状况。受体 子通道蛋白酸化过程及其调节机制对于分析细胞信号转导的过程及细胞功能有着重要的作用。     

百日咳杆菌69KDa外膜蛋白的分离纯化及生物学特性研究

本文发展了一种从百日咳杆菌Ⅰ相菌株中纯化６９ＫＤａ外膜蛋白的简易方法,将细菌体经加热浸提、乙醇沉淀蛋白、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析精制而成。用ＳＤＳ－ＰＡＧＥ、免疫印迹、光密度仪扫描分析,证明纯化制剂为均一的、特异性的６９ＫＤａ外膜蛋白,其收率为５４．２％,纯度达９９．２％,每微克６９ＫＤａ蛋白制剂中的内毒素含量低于０．８５ＥＵ;ＰＴ残留量小于０．１０５ｎｇ。抗６９ＫＤａ蛋白抗血清能     

In vivo phosphorylation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases

2,3-cyclic nucleotide 3-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [-³²]ATP with cAMP (5 M) and cAMP (5 M) + catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that is is likely a PKA site.     

Characterization of Infant Rat Cerebral Cortical Membrane Proteins Phosphorylated In Vivo: Identification of the ACTH-Sensitive Phosphoprotein B-50

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.     

Protein and nuclear changes in pig eggs at fertilization.

The nuclear restructuring that occurs between insemination and full pronuclear formation in pig eggs is accompanied by posttranslational changes to specific egg proteins. Sperm penetration begins in vitro at 3 hr postinsemination (hpi). By 5 hr, decondensing sperm heads and anaphase II plates are observed in 50% of eggs, and, by 8 hpi, both male and female pronuclei have formed. Three consistent changes to the pattern of newly synthesised proteins are triggered in this period; they affect the 46K, 25K, and 22K polypeptides. Changes are also triggered in the 180-200K polypeptides and in the 14K polypeptides, but these are highly variable. The same changes in the prefertilization pattern were observed when prelabelled eggs were used and new protein synthesis was suppressed. The first and most abrupt change involves the apparent catabolic elimination of a group of 46K unphosphorylated polypeptides (pl 7.3-6.4), whose synthesis was greatest before germinal vesicle breakdown but declined slowly in the final phase of maturation, then declined precipitously after activation. Ageing (beyond maturation) also leads to the disappearance of these polypeptides. The progressive disappearance of a set of 25K polypeptides and the concomitant appearance of a dominant 22K polypeptide is the most characteristic fertilization-induced modification to porcine egg proteins. These modifications begin within 1 hr of sperm penetration or activation, are specific to the pig, and involve heavily phosphorylated polypeptides (25K, pl 6.7-6.0) whose synthesis is begun in the early metaphase I stage. Dual ([35S] and [32P]) labelling, protein blocking experiments, and use of alkaline phosphatase suggest that dephosphorylation selectively affects these 25K polypeptides and is mainly or wholly responsible for converting them (completely within 6 hr) to a single, new (22K, pl 7.6) species that is positively charged. The 25K/22K polypeptide modification has a close temporal relationship with the formation of the male and female pronuclei.     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

Exhaustive exercise affects fluidity and alpha-tocopherol levels in brain synaptosomal membranes of normal and vitamin E supplemented rats

Alpha-tocopherol level and fluidity were studied in the neuronal membrane of rat brain after exhaustive exercise. The order parameter, 5-doxyl-stearic acid (5-DS), which is utilized for assessing the fluidity of the lipid bilayer closer to the hydrophilic face of the membrane, decreased in the pons-medulla oblongata, and the motion parameter, 16-doxyl-stearic acid (16-DS) for the core of the lipid bilayer, decreased in the cortex, hippocampus, hypothalamus and striatum, whereas it increased in the cerebellum after exercise. The w/s ratio of n-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimido (maleimido-TEMPO) for the conformation of SH-protein also decreased in the hippocampus and midbrain after exercise. These changes were not observed in alpha-tocopheryl acetate supplemented rats after exercise. Although the levels of 5-DS, 16-DS and maleimido-TEMPO were affected by alpha-tocopheryl acetate in rat neuronal membranes, fluidity changes were reversible with exercise.     

Staurosporine Activates a 60,000 Mr Protein Kinase in Bovine Chromaffin Cells That Phosphorylates Myelin Basic Protein In Vitro

Abstract: Bovine chromaffin cells contain a family of renaturable protein kinases. One of these, a 60,000 M_r kinase (PK60) that phosphorylated myelin basic protein in vitro, was activated fourfold when cells were treated with the protein kinase inhibitor Staurosporine. Because staurosporine inhibits protein kinase C, the role of this kinase in the regulation of PK60 activity was investigated. Fifty nanomolar Staurosporine produced half-maximal inhibition of protein kinase C activity in chromaffin cells, whereas ∼225 n M Staurosporine was required to induce half-maximal activation of PK60. Other protein kinase C inhibitors, H-7 and K-252a, did not mimic the effect of Staurosporine on PK60 activity. Chromaffin cells have three protein kinase C isoforms: α, ε, and ζ. Prolonged treatment with phorbol esters depleted the cells of protein kinase C α and ε, but not ζ. Neither activation nor depletion of protein kinase C affected the basal activity of PK60. Moreover, Staurosporine activated PK60 in cells depleted of protein kinase C α and e; thus, Staurosporine appeared to activate PK60 by a mechanism that does not require these protein kinase C isoforms. Incubation of cell extracts with Staurosporine in vitro did not activate PK60. Incubation of these extracts with adenosine 5'-O-(3-thiotriphosphate), however, caused a twofold activation of PK60. Although this suggests that PK60 activity is regulated by phosphorylation, the mechanism by which Staurosporine activates PK60 is not known. Staurosporine has been reported to promote neurite outgrowth from chromaffin cells. The role of PK60 in mediating the effects of Staurosporine on chromaffin cell function remains to be determined.     

The plasma-membrane H+-ATPase from beet root is inhibited by a calcium-dependent phosphorylation

Several plasma-membrane proteins from beet root (Beta vulgaris L.) have been functionally incorporated into reconstituted proteoliposomes. These showed H⁺-ATPase activity, measured both as ATP hydrolysis and H⁺ transport. The proton-transport specific activity was 10 times higher than in plasma membranes, and was greatly stimulated by potassium and valinomycin. These proteoliposomes also showed calcium-regulated protein kinase activity. This kinase activity is probably due to a calmodulin-like domain protein kinase (CDPK), since two protein bands were recognized by antibodies against soybean and Arabidopsis CDPK. This kinase phosphorylated histone and syntide-2 in a Ca²⁺-dependent manner. Among the plasma-membrane proteins phosphorylated by this kinase, was the H⁺-ATPase. When the H⁺-ATPase was either prephosphorylated or assayed in the presence of Ca²⁺, both the ATP-hydrolysis and the proton-transport activities were slower. This inhibition was reversed by an alkaline-phosphatase treatment. A trypsin treatment (that has been reported to remove the C-terminal autoinhibitory domain from the H⁺-ATPase) also reversed the inhibition caused by phosphorylation. These results indicate that a Ca²⁺-dependent phosphorylation, probably caused by a CDPK, inhibits the H⁺-ATPase activities. The substrate of this regulatory phosphorylation could be the H⁺-ATPase itself, or a different protein influencing the ATPase activities. Received: 1 May 1997 / Accepted: 25 June 1997     

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.