核糖体S6蛋白激酶p90rsk与卵母细胞减数分裂

丝裂原活化蛋白激酶（MAPK）信号途径对减数分裂有重要调节作用,p90^rsk是迄今研究最清楚的MAPK下游靶分子,介导MAPK途径在卵母细胞减数分裂中的多种功能,包括卵母细胞减数分裂的启动、MⅠ/MⅡ期转化和MⅡ期阻滞的维持等.p90^rsk的磷酸化是MAPK激活的结果,而细胞退出减数分裂时,p90^rsk的去磷酸化也发生在MAPK失活以后.介绍了在卵母细胞中p90^rsk的研究进展.     

利用金属离子(Fe3+)配体层析亲和筛选蛋白激酶识别的底物序列

将cAMP依赖的蛋白激酶(cAPK)识别的特征底物序列与噬菌体外膜蛋白g3p融合,展示于线状噬菌体fd的表面,构建了cAPK底物(PKS)噬菌体.噬菌体体外磷酸化标记结果表明,PKS噬菌体上分子量约为60ku的蛋白可被明显磷酸化标记,与PKS-g3p融合蛋白的分子量一致.利用金属离子(Fe³⁺)配体树脂亲和筛选经cAPK磷酸化标记的Phage display随机15肽库,4轮筛选后挑取单克隆进行DNA序列测定,确定展示于噬菌体表面的多肽序列.结果表明,在所挑选的14个克隆中有5个克隆具有典型的cAPK磷酸化序列特征(R)RXS/T.对这些噬菌体的体外磷酸化标记实验结果显示,其中有(R)RXS/T序列特征的噬菌体可在分子量约60ku处被特征性磷酸化标记,与PKS噬菌体的磷酸化标记特征一致;其他有一些不具备(R)RXS/T序列特征的噬菌体也可被特征性磷酸化.     

钙调素及其结合蛋白BP-10对类囊体膜蛋白磷酸化的作用

报道光诱导的内源类囊体膜蛋白的磷酸化可被一种新的植物钙调素(Calmodulin ,CaM )结合蛋白BP 1 0 (CaMBP -1 0 )显著抑制 ,并且抑制作用能被外加CaM消除 .同时 ,此磷酸化反应也可被EGTA和CaM拮抗剂TFP(trifluoperazine)及W 7(N ( 6 aminohexyl) -5- chloro -1 naphthalenesulfonamide)抑制 .提示 :( 1 )Ca ²⁺和CaM可能参与并调节植物光合作用 ;( 2 )催化类囊体膜蛋白磷酸化的激酶可能受Ca ²⁺和CaM调控 .进一步实验表明BP -1 0对类囊体膜蛋白的脱磷酸化作用无任何影响 .     

组蛋白乙酰化/去乙酰化与基因表达调控

组蛋白是真核生物染色质的主要成分,组蛋白修饰(如甲基化、乙酰化、磷酸化、泛素化等)在真核生物基因表达调控中发挥着重要的作用.在这些修饰中,组蛋白乙酰化/去乙酰化尤为重要.组蛋白乙酰化/去乙酰化可通过改变染色质周围电荷或参与染色质构型重建而影响基因表达;更重要的是组蛋白乙酰化/去乙酰化可形成一种特殊的“密码”,被其它蛋白质识别,影响多种蛋白质因子的活动或与其相互作用,参与到基因表达调控的整个网络中.     

磷酸化和脱磷酸化对牛脑63kD环核苷酸磷酸二酯酶同工酶活性的调节

本文报道了CaM依赖性磷酸化和脱磷酸化对牛脑63kD PDE同工酶活性的调节作用。实验结果如下:(1)在存在Ca²⁺和CaM时,提纯的牛脑Ca²⁺/CaM-PK Ⅱ能催化牛脑63kD PDE同工酶磷酸化。最大磷酸参入量是1mol/mo1亚基;(2)在Ni²⁺和CaM存在时,提纯的牛脑钙调神经磷酸酶能催化磷酸化型63kDPDE同工酶脱磷酸化;(3)CaH²⁺对磷酸化型63kD PDE同工酶的半激活浓度(AC₅₀)高于非磷酸化型。     

乙醛对人类神经tau磷酸化的影响

用乙醛对人类神经tau进行醛胺化,通过NCLK(neuronal cdc2-like protein kinase)和［γ-³²P］ATP对其磷酸化.磷酸化的产物经胃蛋白酶降解及HPLC(C-18)分析降解片段,发现醛胺化tau的降解物中有两个新的磷酸化肽段(A4和A6).     

ASK1激活的分子机制与相关疾病

凋亡信号调节激酶1(Apoptosis signal-regulating kinase 1,ASK1)是细胞丝裂原活化蛋白激酶激酶激酶(mitogen-activated protein kinase kinase kinase,MAP3Ks)家族成员之一,在调节细胞凋亡过程中起到非常重要的作用.在正常细胞中,ASK1的活化受到严格的控制,如苏氨酸/丝氨酸磷酸化和去磷酸化、蛋白-蛋白相互作用等.多种应激和促炎因子能激活ASK1,因此在多种生理和病理过程中都有活化的ASK1的参与.     

心肌肌钙蛋白Ⅰ的磷酸化与非磷酸化调节

由于心肌肌钙蛋白复合体Ⅰ亚基(Troponin Ⅰ,TnⅠ)特殊的分子结构,使其在心肌收缩过程中起"分子开关"的重要作用.心肌TnⅠ具有6个磷酸化位点,第23/24位丝氨酸残基可被蛋白激酶A(PKA)、蛋白激酶D(PKD)和蛋白激酶G(PKG)磷酸化,发挥正性肌力作用;第43/45位丝氨酸残基以及第144位酪氨酸残基可被蛋白激酶C(PKC)磷酸化,可能主要起负性肌力作用;蛋白激活激酶(PAK)磷酸化第149位丝氨酸残基后的作用尚待探明.另外,经蛋白水解酶calpain降解含磷酸化位点的片段,产生去磷酸化作用;亦可通过降解一些特定片段来改变TnⅠ空间构象,引起非磷酸化调节作用.     

神经节苷脂GM3对J6－2细胞蛋白质磷酸化的影响

通过研究神经节苷脂GM₃对国人单核样白血病细胞系J6-2细胞蛋白质磷酸化的影响,在[γ- ³²P]ATP,GM₃,ATP,Mg²⁺与J6-2细胞液及颗粒两部分共同反应,10min(30℃)体系中,观察到GM₃对两部分蛋白质磷酸化的调节作用.GM₃(100μmol/L)促进颗粒部分分子量为180 000,87 000,78 000,67 000,43 000及31 000的蛋白质磷酸化,促进胞液部分分子量为87 000及56 000的蛋白质磷酸化,而且能抑制70 000及43 000蛋白质磷酸化.由于GM₃已被前人证实能对J6-2细胞起分化作用,其作用时间长达4-6d,很可能GM₃对蛋白质磷酸化作用的调节是GM₃促分化作用的早期信号.     

基于Ti4+-IMAC富集的分枝菌酸小杆菌深度覆盖磷酸化蛋白质组研究

【目的】本研究以分枝菌酸小杆菌(Mycolicibacterium smegmatis)为研究对象,探索适于原核微生物理想的磷酸化富集方法。【方法】我们比较了二氧化钛(TiO₂)、Fe³⁺-NTA和Ti⁴⁺螯合在磷酸酯修饰的固相微球(Ti⁴⁺-IMAC) 3种不同富集方法磷酸化肽段的富集效率,并用不同分辨率的质谱仪评估富集稳定性。【结果】Ti⁴⁺-IMAC富集效率最高,磷酸化位点数是TiO₂或Fe³⁺-NTA方法的7倍以上;TiO₂和Fe³⁺-NTA方法富集到的磷酸化位点数相差不大,与已报道的用TiO₂方法富集的磷酸化位点数目接近。Ti⁴⁺-IMAC富集结果稳定性很好,高分辨率Lumos质谱仪鉴定到的磷酸化位点数是Velos的2.6倍。【结论】本研究较高效地实现了分枝菌酸小杆菌磷酸化事件的鉴定,共鉴定到2 280个磷酸化蛋白、10 880个磷酸化肽段及4 433个可信磷酸化位点,有望用于其他微生物的磷酸化蛋白质组学研究。     

Phosphorylation of the 24p3 protein secreted from mouse uterus in vitro and in vivo

The 24p3 protein is a 25 KDa glycoprotein, having been purified from mouse uterine fluid. Thr⁵⁴, Ser⁸⁸, and Thr¹²⁸/Ser¹²⁹ on the protein molecule were predicted to be the phosphorylation site of casein kinase II, protein kinase C, and cAMP-dependent protein kinase, respectively. Incorporation of phosphate to this protein from [-³²P]-ATP was tested in the solution suitable for the three kinases. Neither casein kinase II nor cAMP-dependent protein kinase reacted to the 24p3 protein; however, protein kinase C demonstrated phosphorylation to this protein. This phosphorylation may be competing with a polypeptide segment: Arg⁷⁹-Tyr-Trp-Ilu-Arg-Thr-Phe-Val-Pro-Ser⁸⁸-Ser-Arg-Ala-Gly-Gln-Phe-Thr-Leu-Gly⁹⁷ in the 24p3 protein molecule. To support this theory, Ser⁸⁸ is a phosphorylation site of protein kinase C on 24p3 protein. The enzyme kinetic parameter, based on the Michaelis-Menten equation, determined Km to be 2.96 M in the phosphorylation of 24p3 protein by the kinase. Both of the phosphorylated and dephosphorylated form of 24p3 protein can enhance the cAMP-dependent protein kinase activity in vitro. In addition, this experiment will show for the first time that serine-phosphorylated 24p3 protein exists in mouse uterine tissue.     

Calcium/calmodulin-dependent protein kinase II: role in learning and memory

Numerous studies over the past decade have established a role(s) for protein phosphorylation in modulation of synaptic efficiency. This article reviews this data and focuses on putative functions of Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) which is highly concentrated at these synapses which utilize glutamate as the neurotransmitter. Evidence is presented that CaM-kinase II can phosphorylate these glutamate receptor/ion channels and enhance the ion current flowing through them. This may contribute to mechanisms of synaptic plasticity that are important in cellular paradigms of learning and memory such as long-term potentiation in the hippocampus.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Identification of mitogen-activated protein kinase-activated protein kinase-2 as a vimentin kinase activated by okadaic acid in 9L rat brain tumor cells

Organization of intermediate filament, a major component of cytoskeleton, is regulated by protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved protein kinases and phosphatases. Blocking dephosphorylation by protein phosphatase inhibitors such as okadaic acid (OA) leads to an apparent activation of protein kinase(s) and to genuine activation of phosphatase-regulated protein kinase(s). Treatment of 9L rat brain tumor cells with OA results in a drastically increased phosphorylation of vimentin, an intermediate filament protein. In-gel renaturing assays and in vitro kinase assays using vimentin as the exogenous substrate indicate that certain protein kinase(s) is activated in OA-treated cells. With specific protein kinase inhibitors, we show the possible involvement of the cdc2 kinase- and p38 mitogen-activated protein kinase (p38^MAPK)-mediated pathways in this process. Subsequent in vitro assays demonstrate that vimentin may serve as an excellent substrate for MAPK-activated protein kinase-2 (MAPKAPK-2), the downstream effector of p38^MAPK, and that MAPKAPK-2 is activated with OA treatment. Comparative analysis of tryptic phosphopeptide maps also indicates that corresponding phosphopeptides emerged in vimentin from OA-treated cells and were phosphorylated by MAPKAPK-2. Taken together, the results clearly demonstrate that MAPKAPK-2 may function as a vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38^MAPK signaling cascade, via MAPKAPK-2, in the maintenance of integrity and possible physiological regulation of intermediate filaments. J. Cell. Biochem. 71:169–181, 1998. © 1998 Wiley-Liss, Inc.     

Studies on the possible role of protein phosphorylation in the transduction of the ethylene signal

Previous work in our laboratory has demonstrated the existence of high affinity binding sites for the plant growth regulator ethylene. The ethylene binding protein (EBP), from Phaseolus cotyledons, shows many of the characteristics of a functional receptor for ethylene, has been purified on SDS-PAGE and polyclonal antibodies raised in rabbits. Current work involves the investigation of the ethylene transduction signal in a number of ethylene-responsive tissues.In peas, it has been shown that ethylene promotes the phosphorylation of specific proteins of similar molecular weight to the EBP from Phaseolus. Such ethylene-induced phosphorylation can be inhibited by the ethylene antagonist, 2,5-NBD. The antibodies raised to the EBP from Phaseolus have been shown to immunoprecipitate ³²P-labelled proteins from membrane protein preparations obtained from pea tissue. Studies on ethylene binding in pea have also shown that the binding of ethylene may be regulated by phosphorylation. Thus, under conditions which promote phosphorylation, binding is inhibited, whereas the reverse is true under conditions which enhance dephosphorylation.Further work is described which examines the effect of protein kinase, protein phosphatase and calcium channel inhibitors on ethylene-induced phosphorylation in peas, together with wild-type (WT) and ethylene insensitive (eti) mutants of Arabidopsis thaliana. The effects of these treatments can be monitored in vivo using the ethylene-induced triple response as a screen. Furthermore, the protein profiles of such treated seedlings can then be compared by labelling protein extracts with ³²P and subjecting the samples to SDS-PAGE followed by autoradiography.     

Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs.     

Redox dependence of the blue-light-induced phosphorylation of a 100-kDa protein on isolated plasma membranes from tips of coleoptiles

A blue-light-induced rapid phosphorylation of a 100-kDa protein localized in plasma membranes of phototropically sensitive tips of maize (Zea mays L.) coleoptiles was studied. Since, under in-vivo conditions or in a crude homogenate of tips, cytosolic ATP is the phosphate donor for the light-induced phosphorylation of this protein, a subsequent in-vitro phosphorylation by [³²P]ATP is prevented. However, in-vitro irradiation of microsomal membranes isolated from non-irradiated tips followed by a 1-min incubation with [³²P]ATP resulted in a strong phosphorylation (labelling) of the 100-kDa plasma-membrane protein. This process was saturated by a 7-s light pulse (200 μmol photons·m^?2·s^?1). In the absence of [³²P]ATP the capacity for in-vitro phosphorylation of the 100-kDa protein after a 30-s light pulse declined slowly within 60 min but could be reconstituted by a new light pulse in the presence of reducing compounds. Moreover, when plasma membranes which had been stored frozen were used, reducing compounds such as NADH, NADPH, ascorbate, glutathione or dithiotreithol enhanced the light-triggered in-vitro phosphorylation. These compounds were unable to elicit or enhance the phosphorylation in the dark. It is suggested that the transfer of (blue-light) excited electrons from the chromophore moiety of the receptor to the target (either the 100-kDa protein or the protein kinase itself) is facilitated when reducing compounds instantly eliminate the positive charge generated at the chromophore. The transferred electrons could finally alter the redox state and-or the conformation of either the 100-kDa protein, rendering it susceptible to the action of a protein kinase, or the protein kinase which would then be capable of phospho-rylating the 100-kDa protein.     

Nature of the intrinsic protein kinases involved in phosphorylation of non-histone proteins in intact prostatic nuclei: further identification of androgen-sensitive protein kinase reactions

Nuclei isolated from rat ventral prostate contain a number of messenger-dependent and -independent protein kinases. Studies were undertaken to determine the relative contribution of these protein kinases in phosphorylation of non-histone proteins (NHPs) in isolated nuclei. The data suggest that messenger-dependent protein kinases such as those dependent on cAMP or Ca²⁺/calmodulin or Ca^2–/phospholipid may be present in very small amounts in intact isolated nuclei, and thus appear not to be significantly involved in phosphorylation of endogenous NHPs. Messenger-independent nuclear associated protein kinases PK-N1 and PK-N2 are known to catalyze the phosphorylation of NHPs in vitro (Goueli SA, et al., Eur J Biochem 113: 45–51, 1980). Of these, the intrinsic heparin-sensitive PK-N2 as compared with heparin-insensitive PK-N1 appeared to be the predominant protein kinase engaged in phosphorylation of NHPs in intact nuclei. About 78–88% of NHP phosphorylation in intact nuclei was inhibited by heparin suggesting that the remaining 12–22% phosphorylation of NHPs was catalyzed via the heparin-insensitive protein kinase(s). Further, the data provide additional evidence that heparin-sensitive PK-N2 is the one that is most responsive to androgenic status in the animal.Abbreviations NHP Non-Histone Protein - PMSF Phenylmethylsulfonyl Fluoride - DTT Dithiothreitol - SDS Sodium Dodecyl Sulfate     

蛋白质棕榈酰化对离子型谷氨酸受体运输的调节作用

棕榈酰化是一种可逆的翻译后修饰,其对蛋白质的定位和功能具有重要的调节意义.离子型谷氨酸受体有N-甲基-D-天冬氨酸(NMDA)受体、α-氨基羟甲基恶唑丙酸(AMPA)受体和人海藻酸受体.近期研究发现,它们的棕榈酰化修饰对其膜表面分布和内化均具有重要的意义.其中NMDA受体在其C末端有2个不同的棕榈酰化位点.1个位于C末端近膜区(CysclusterⅠ),它的棕榈酰化可以增高酪氨酸的磷酸化水平,增加受体膜表面分布,影响神经元中NMDA受体的组构性内化;另1个位于C末端中部(CysclusterⅡ),它受到蛋白质酰基转移酶GODZ的调节,使得受体在高尔基体大量积聚,从而影响受体的膜表面分布.与NMDA受体相似,AMPA受体也存在2个棕榈酰化位点.1个位于在第2跨膜域,受蛋白质酰基转移酶GODZ的调节,能导致AMPA受体在高尔基体的积聚.另1个位点在受体C末端近膜区,它的棕榈酰化能降低AMPA受体和4.1N蛋白的相互作用,并调节受体的内化.这两种离子型谷氨酸受体在棕榈酰化机制上虽然存在差异,但均对受体的运输、膜表面分布和内化具有十分重要的作用.