Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex

Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. We show that in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation.     

Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta)

Glucose homeostasis depends on insulin responsiveness in target tissues, most importantly, muscle and liver. The critical initial steps in insulin action include phosphorylation of scaffolding proteins and activation of phosphatidylinositol 3-kinase. These early events lead to activation of the serine-threonine protein kinase Akt, also known as protein kinase B. We show that mice deficient in Akt2 are impaired in the ability of insulin to lower blood glucose because of defects in the action of the hormone on liver and skeletal muscle. These data establish Akt2 as an essential gene in the maintenance of normal glucose homeostasis.     

Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane

Brassinosteroids, the steroid hormones of plants, are perceived at the plasma membrane by a leucine-rich repeat receptor serine/threonine kinase called BRI1. We report a BRI1-interacting protein, BKI1, which is a negative regulator of brassinosteroid signaling. Brassinosteroids cause the rapid dissociation of BKI1-yellow fluorescent protein from the plasma membrane in a process that is dependent on BRI1-kinase. BKI1 is a substrate of BRI1 kinase and limits the interaction of BRI1 with its proposed coreceptor, BAK1, suggesting that BKI1 prevents the activation of BRI1.     

高粱根质膜的纯化和膜蛋白的提取

利用高粱根作为实验材料,用不连续蔗糖密度梯度和Dextran T-500/PEG-3350两相法探讨了纯化质膜的过程,以及去垢剂对K+运输蛋白的增溶效率.结果表明,由6 mL 40%(W/W),4 mL 34%(W/W)和4 mL 22%(W/W)蔗糖组成的不连续蔗糖梯度,经80 000×g离心3 h后,在34%～40%界面处富含质膜.在质膜K+运输蛋白的增溶效果上,6.5 mmol/L CHAPS最为理想.     

蛋白激酶B短发夹环RNA表达载体的构建及其对血管平滑肌细胞增殖活性的影响

目的构建靶向蛋白激酶B基因的短发夹环RNA表达载体,观察其对血管平滑肌细胞增殖活性的影响。方法设计多个针对大鼠蛋白激酶B基因的短发夹环RNA序列,化学合成方法合成并经pGEM-T载体克隆后双酶切,将cDNA序列插入逆转录病毒载体pLXIN,包装后获得蛋白激酶B的逆转录表达载体,感染血管平滑肌细胞,Northern blot和Western blot法检测蛋白激酶B及其下游底物的表达变化,流式细胞仪检测细胞周期变化,MTT法检测血管平滑肌细胞增殖活性的改变。结果成功构建蛋白激酶B基因的逆转录病毒载体并包装,感染血管平滑肌细胞,证实其能显著抑制蛋白激酶B的mRNA和蛋白产物表达,下游的p70s6k表达相应减少;被感染血管平滑肌细胞的分裂、增殖过程受阻,更多细胞停滞在G0/G1期。结论成功构建蛋白激酶B基因逆转录病毒RNA干扰表达载体,感染血管平滑肌细胞能够明显抑制其分裂、分化和增殖。     

Living with lethal PIP3 levels: viability of flies lacking PTEN restored by a PH domain mutation in Akt/PKB

The phosphoinositide phosphatase PTEN is mutated in many human cancers. Although the role of PTEN has been studied extensively, the relative contributions of its numerous potential downstream effectors to deregulated growth and tumorigenesis remain uncertain. We provide genetic evidence in Drosophila melanogaster for the paramount importance of the protein kinase Akt [also called protein kinase B (PKB)] in mediating the effects of increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations that are caused by the loss of PTEN function. A mutation in the pleckstrin homology (PH) domain of Akt that reduces its affinity for PIP3 sufficed to rescue the lethality of flies devoid of PTEN activity. Thus, Akt appears to be the only critical target activated by increased PIP3 concentrations in Drosophila.     

Bacteriophage M13 procoat protein inserts into the plasma membrane as a loop structure

The major coat protein of bacteriophage M13 is synthesized as a precursor, the procoat, with a typical leader (signal) sequence of 23 residues at its NH2-terminus. A fusion protein that contains the NH2-terminal 141 residues of cytoplasmic ribulokinase and all but the first ten residues of M13 procoat was made. The fusion protein inserts into the plasma membrane of Escherichia coli and is processed by leader peptidase to give rise to a leader peptide of 155 residues and the mature coat protein of 50 residues. The NH2-terminus of the leader peptide remains in the cytoplasm and is protected from protease added to the medium outside of the cell. This indicates that M13 procoat inserts into the membrane as a loop structure and that the NH2-terminus of a leader peptide remains within the cytoplasm during membrane insertion.     

Phosphorylation and regulation of Raf by Akt (protein kinase B)

Activation of the protein kinase Raf can lead to opposing cellular responses such as proliferation, growth arrest, apoptosis, or differentiation. Akt (protein kinase B), a member of a different signaling pathway that also regulates these responses, interacted with Raf and phosphorylated this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibited activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt.     

Vesicle formation at the plasma membrane and trans-Golgi network: the same but different

An elaborate vesicle transport system supports the active exchange of membranes and protein cargo between the plasma membrane and the trans-Golgi network. Many observations suggest that highly conserved mechanisms are used in vesicle formation and scission. Such similarity is found both at the level of the receptor-ligand sequestration process that uses clathrin and associated polymeric and monomeric adaptor proteins, and in the machinery used to deform and vesiculate lipid membranes.     

山核桃水通道蛋白CcPIP同源基因克隆与表达分析

利用RACE方法,在山核桃嫁接过程中扩增出山核桃水通道蛋白同源基因CcPIP。应用生物信息学软件进行分析,预测该序列编码294个氨基酸,具有6个跨膜区,有MIP家族信号序列和高等植物PIP高度保守序列。通过NCBI同源性比较分析表明,该基因与葡萄、菜豆等物种的水通道蛋白基因同源性达到99%。荧光定量RT-PCR分析表明,CcPIP基因在芽中的表达量最低,其次是雄花序,果实,幼叶和茎,而在根中表达量最高。在山核桃嫁接前后,CcPIP基因在接穗中表达趋势和在砧木中的表达一致。CcPIP基因的表达量在山核桃嫁接前砧木和接穗中都有强烈表达,但在嫁接后3 d急剧下降,分别在砧木和接穗中下降了5倍和2倍。在随后的11 d里,CcPIP基因的表达量在砧木和接穗中开始上升,至嫁接后14 d基因的转录水平比嫁接后3 d分别增加了7倍和4倍。该CcPIP基因参与山核桃嫁接成活的水分运输过程中起到基因表达调控的作用。     

Anatomy and dynamics of a supramolecular membrane protein cluster

Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules. Self-association depends on weak homophilic protein-protein interactions. Simulations suggest that clustering immobilizes and conformationally constrains the molecules. Moreover, a balance between self-association and crowding-induced steric repulsions is sufficient to explain both the size and dynamics of syntaxin clusters and likely of many oligomerizing membrane proteins that form supramolecular structures.     

Comment on "A G protein coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid"

Liu et al. (Reports, 23 March 2007, p. 1712) reported that the Arabidopsis thaliana gene GCR2 encodes a seven-transmembrane, G protein-coupled receptor for abscisic acid. We argue that GCR2 is not likely to be a transmembrane protein nor a G protein-coupled receptor. Instead, GCR2 is most likely a plant homolog of bacterial lanthionine synthetases.     

Selective solubilization of a protein component of the red cell membrane

Approximately 20 percent of the membrane-bound protein of erythrocyte ghosts can be solubilized and obtained free of other membrane components by dialysis against adenosine triphosphate and 2-mercaptoethanol. This protein forms one major band on polyacrylamide gels and a single boundary in free-boundary electrophoresis, and it undergoes polymerization in the presence of divalent cations to form coiled filaments visible by electron microscopy. Antibodies to this membrane protein react specifically with red blood cells or their membrane ghosts but do not react with serum, erythrocyte cytoplasm, or other blood cells. The functional role of this protein is unknown, but it appears to be involved in maintaiining the structure of the red cell membrane. We suggest that this protein be called Spectrin since it is obtained from membrane ghosts.     

Clathrin: a role in the intracellular retention of a Golgi membrane protein

Yeast mutants deficient in the clathrin heavy chain secrete a precursor form of the alpha-factor, a peptide-mating pheromone. Analysis of this defect indicates that the endoprotease Kex2p, which is responsible for initiating proteolytic maturation of the alpha-factor precursor in the Golgi apparatus, is unexpectedly present at the plasma membrane in mutant cells. This result suggest that clathrin is required for the retention of Kex2p in the Golgi apparatus.     

Mobility and the restriction of mobility of plasma membrane lectin-binding components

Labeling by ferritin-conjugated agglutinins from Ricinus communis was used to demonstrate the relative mobilities of the agglutinin receptors located in specific regions on plasma membranes of rabbit spermatozoa. The relative mobility of lectin receptors was higher on postacrosomal regions of sperm than on acrosomal and tail regions. Lectin-induced clustering could not be demonstrated in the acrosomal and tail regions, an indication of the existence of localized restraints on the mobilities of lectin receptors. A system of transmembrane restraints may maintain the segregation of plasma membrane components into membrane domains on certain highly differentiated cells.     

番茄质膜H+-ATPase家族基因的鉴定和表达分析

植物质膜H+-ATPase（Ec3.6.1.3）是一类普遍存在于细胞质膜上通过水解三磷酸腺苷（ATP）产生能量,将细胞质中的氢离子（H+）逆浓度泵出细胞的运输蛋白。植物中的质膜H+-ATPase由一个多基因家族所编码,其功能涉及到植物生长发育的多个生理过程。通过对全基因组检索在茄科Solanaceae植物番茄Solanum lycopersicum中共鉴定到8个编码质膜H+-ATPase的同源基因（LHA1~8）。生物信息学分析显示:这8个LHA基因具有较高的序列相似性和较为保守的外显子/内含子结构特征。实时荧光定量聚合酶链式反应（qRT-PCR）分析显示,LHA1~4在所有被检测的组织器官中都有表达,LHA5~7几乎只在花器官中高量表达,而LHA8在正常培养和养分（氮、磷、钾和镁）缺乏以及高盐胁迫处理条件下几乎都不表达,但能够在被菌根真菌侵染的根系中强烈表达。将一段2 669 bp的LHA8的启动子融合GUS报告基因转入到烟草Nicotiana tabacum中发现,GUS基因几乎只在被菌根真菌菌丝侵入形成丛枝的根系细胞中特异性表达。图 5 表 2 参22     

不同氮素营养形态对香蕉生长及其根系质子泵活性的影响

[目的]研究不同氮素营养形态(不同比例NH4+/NO3-)对香蕉生长及根系质子泵活性的影响.[方法]在温室中水培巴西种香蕉(Musa AAA Cavendish cv.Baxi),设置5个处理:100％硝态氮处理(100％N);75％硝态氮和25％铵态氮处理(75％N+25％A);50％硝态氮和50％铵态氮处理(50％N+50％A);25％硝态氮和75％铵态氮处理(25％N+75％A);100％铵态氮处理(100％A).测定香蕉的生物量和N、P、K、Ca、Mg养分含量,并分离根系细胞膜,测定质子泵活性,建立植物生长状况与质子泵活性之间的联系.[结果]铵硝混合营养比单一的铵态氮或硝态氮营养对香蕉生长具有更好的生长效果,其中以50％硝态氮+50％铵态氮处理中香蕉植株的生物量最大,植物体内养分含量也最高;香蕉根际pH值与不同比例NH4+/NO3-有关,在100％铵态氮处理时最低,随着硝态氮比例的增加,pH值逐渐上升;香蕉根系细胞膜质子泵活性在100％铵态氮处理时最高,随着硝态氮比例的增加,质子泵活性逐渐降低.[结论]质子泵活性既与营养液中的不同比例NH4/NO3有关,也受到铵态氮或硝态氮吸收后根际pH值变化的影响.质子泵活性过高与过低都是植物生长受到外界胁迫后的一种应答,或是总体生长状况不良的一种反应.相反,质子泵活性处于一个相对稳定的适中状态才说明植物处于一个最佳的生长状态.     

Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane

The quaternary structure and functional properties of synaptophysin, a major integral membrane protein of small presynaptic vesicles, were investigated. Cross-linking and sedimentation studies indicate that synaptophysin is a hexameric homo-oligomer, which in electron micrographs exhibits structural features common to channel-forming proteins. On reconstitution into planar lipid bilayers, purified synaptophysin displays voltage-sensitive channel activity with an average conductance of about 150 picosiemens. Because specific channels and fusion pores have been implicated in vesicular uptake and release of secretory compounds, synaptophysin may have a role in these processes.     

猪流产嗜性衣原体MOMP基因的克隆及原核表达

【目的】克隆猪流产嗜性衣原体青海株主要外膜蛋白(Major outer membrane protein,MOMP)基因,并进行序列分析及原核表达。【方法】根据GenBank公布的猪流产嗜性衣原体MOMP基因的核苷酸序列,设计并合成4条特异性引物,用套式PCR方法扩增猪流产嗜性衣原体青海株MOMP基因,将其克隆入pMD18-T载体中,进行测序及序列分析。然后将MOMP基因亚克隆入原核表达载体pGEX4T-1中,在大肠杆菌BL21(DE3)中用IPTG诱导表达,对表达产物进行SDS-PAGE和Western blot检测。【结果】扩增到猪流产嗜性衣原体青海株1170bp的MOMP全长基因。序列分析结果表明,该基因与已发表的猪流产嗜性衣原体B11001株和CP/12株核苷酸同源性均为99.7%。SDS-PAGE电泳可检测到分子质量约为66ku的融合蛋白,主要以包涵体形式存在。Western-blot分析表明,重组蛋白可被猪流产嗜性衣原体抗体识别。【结论】成功克隆了猪流产嗜性衣原体青海株MOMP基因,并进行了原核表达,表达的蛋白具有抗原活性。