酪氨酸蛋白磷酸酶

本文介绍了酪氨酸蛋白磷酸酶的研究现状。对各种组织中纯化的多种酪氨酸蛋白磷酸酶的性质研究,发现在大多数组织和细胞中存在多种形式的该酶,它们可分成三大类,但各种形式的酶之间的相互关系尚不清楚。酪氨酸蛋白磷酸酶在细胞的生长、分化、转化及信号传递过程中可能起重要作用。     

蛋白酪氨酸磷酸酶-PEST在生理调节及相关疾病中的作用

蛋白质分子中酪氨酸残基可逆性的磷酸化是细胞内信号分子传导的基本方式。两类作用相反的酶参与磷酸化的调节:蛋白酪氨酸激酶(protein tyrosinekinase,PTK)和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)。含脯氨酸-谷氨酸-丝氨酸-苏氨酸(P-E-S-T)结构域的蛋白酪氨酸磷酸酶(PTP-PEST)属于非受体型酪氨酸磷酸酶类,其本身能与多种蛋白质相互作用,并在细胞迁移、免疫细胞活化和胚胎发育等生理过程中发挥重要作用。本文对PTP-PEST的结构特点、生理功效、介导的信号传导途径和近年来PTP-PEST在疾病中的作用作一综述。     

糖尿病中蛋白酪氨酸磷酸酶的研究进展

糖尿病是由于胰岛素分泌不足或胰岛素抵抗引起的以血糖升高为特征的代谢性疾病。有研究发现一些蛋白酪氨酸磷酸酶(proteintyrosine phosphatases,PTP)在胰岛素受体信号途径、胰岛素分泌和胰腺β细胞受自身免疫细胞攻击等生理或病理过程中起重要作用。以PTP1B、TCPTP和LYP为代表的PTP通过将底物去磷酸化,拮抗激酶催化的磷酸化反应,在一些信号通路中起到负相调节的作用。在糖尿病患者中发现这些PTP的单核苷酸突变使蛋白表达增加或酶活力增强,因而施用这些潜在靶蛋白的小分子抑制剂成为治疗1型或2型糖尿病可能的新疗法。而PTPIA-2/IA-2β的胞内磷酸酶结构域被发现是大量1型糖尿病患者的自身免疫原,因此可针对PTPIA-2/IA-2β发展早期诊断并预防1型糖尿病的试剂盒。     

蛋白酪氨酸磷酸酶SHP-2在乳腺癌细胞移动及粘附中的作用

探讨蛋白酪氨酸磷酸酶SHP 2在乳腺癌细胞MCF 7的移动及粘附中的作用 .利用基因重组技术分别将野生型SHP 2与突变型SHP 2与绿色荧光蛋白GFP的基因片段构成重组质粒 (SHP 2 GFP、SHP 2C >S GFP) .脂质体转染法分别转入MCF 7中 ,表达成功后筛选并建立SHP 2 GFP和SHP 2C >S GFP细胞株 .荧光显微镜观察细胞移动情况 ,免疫印迹法检测粘附分子E 钙粘蛋白和金属蛋白酶MMP 1及MMP 9的表达 .实验后建立SHP 2 GFP及SHP 2C >S GFP细胞株 ,同时观察到SHP 2C >S GFP细胞的形态发生明显改变 :从梭形状态变成圆形状态 .荧光显微镜发现 ,MCF 7细胞和SHP 2 GFP、SHP 2C >S GFP转染的细胞在 3h、6h、9h的移动情况分别是MCF 7为 10 %、2 3%、5 4% ,SHP 2 GFP为 15 %、4 9%、98% ,SHP 2C >S GFP为 4 %、11%、30 % .免疫印迹结果表明 ,SHP 2C >S GFP细胞的E 钙粘蛋白表达比SHP 2 GFP细胞明显升高 (P <0 0 5 ) .MMP 1及MMP 9的表达量在SHP 2 GFP细胞中有所增强 (P <0 0 5 ) .实验表明 ,SHP 2可能通过调节粘附分子和基质金属磷酸酶而在细胞移动、粘附中发挥重要作用     

蛋白酪氨酸磷酸酶抑制剂的研究进展

蛋白酪氨酸磷酸酶（PTP）作为一类调节细胞活动的重要蛋白,与目前一些常见疾病,如癌症、代谢性疾病、心血管疾病、精神性疾病等密切相关。研究表明,抑制这类蛋白可以有效控制上述相关疾病的发生与发展。文章按不同亚型分类对主要蛋白抑制剂的研究进展进行介绍,以期为蛋白酪氨酸磷酸酶抑制剂的开发提供参考,进一步指导相关药物开发。     

蛋白酪氨酸磷酸酶家族及其生理作用

蛋白酪氨酸磷酸酶（protein tyrosine phosphatase,PTP）催化蛋白质分子中特定位点的磷酸化酪氨酸残基脱磷酸,以＂瀑布式的级联反应＂方式与其他蛋白磷酸酶在细胞内构成调控网络,与蛋白酪氨酸激酶（protein tyrosine kinase,PTK）的作用相反,共同凋节细胞信号转导,在细胞生长、分化、引导有丝分裂、T细胞活化等生理过程中起着重要的作用,尤其在控制细胞磷酸化酪氨酸水平上,蛋白酪氨酸磷酸酶起着高度特异性的积极作用,占据了生导地位.蛋白酪氨酸磷酸酶在人类基因组中主要由90个基因表达,分为4个家族.其催化位点的构象决定了它对可逆的氧化敏感.     

高等植物体内酪氨酸蛋白磷酸酶及其功能

对高等植物体内酪氨酸蛋白磷酸酶及其功能的研究进展作了简要介绍.     

蛋白质酪氨酸磷酸酶与药物靶标

蛋白质酪氨酸磷酸化作用是真核细胞中的一种重要信号作用机制,由蛋白质酪氨酸激酶和蛋白质酪氨酸磷酸酶共同调控.蛋白质酪氨酸磷酸酶在真核细胞代谢进程中起着重要的作用,与许多人类疾病如肿瘤、心血管疾病、免疫缺陷性疾病、传染病、神经性以及代谢方面疾病的发病机制密切相关,许多蛋白质酪氨酸磷酸酶已成为研究和开发治疗人类重大疾病药物的优秀靶标.     

Dephosphorylation of Neurofilaments by Exogenous Phosphatases Has No Effect on Reassembly of Subunits

Exhaustive in vitro dephosphorylation of porcine neurofilaments (NFs) by alkaline or acid phosphatase did not cause a dissociation of the 210-kD (NF-H), 160-kD (NF-M), or 70-kD (NF-L) subunits and had no effect on the reassembly of NFs from urea or guanidine solution. Electron microscopy revealed that the NFs reassembled from isolated or dephosphorylated subunits had similar morphologies. Phosphatase treatment caused significant increases in the mobilities of NF-M and NF-H on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the subunits underwent marked conformational changes after dephosphorylation. Chemical phosphate analysis showed that as isolated NF-H, NF-M, and NF-L contained about 22, 11, and 3 mol phosphate/mol polypeptide, respectively. The corresponding values for the three subunits from alkaline phosphatase-treated NFs were about 8, 6, and 2 mol phosphate/mol polypeptide, respectively. These results indicate the occurrence of a class of phosphate moieties that is not accessible to exogenous phosphatases.     

Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.     

Dimeric Quaternary Structure of Human Laforin

The phosphatase laforin removes phosphate groups from glycogen during biosynthetic activity. Loss-of-function mutations in the gene encoding laforin is the predominant cause of Lafora disease, a fatal form of progressive myoclonic epilepsy. Here, we used hybrid structural methods to determine the molecular architecture of human laforin. We found that laforin adopts a dimeric quaternary structure, topologically similar to the prototypical dual specificity phosphatase VH1. The interface between the laforin carbohydrate-binding module and the dual specificity phosphatase domain generates an intimate substrate-binding crevice that allows for recognition and dephosphorylation of phosphomonoesters of glucose. We identify novel molecular determinants in the laforin active site that help decipher the mechanism of glucan phosphatase activity.     

Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a

Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.     

A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET system for the detection of phosphopeptides using a phosphate-binding tag molecule, Zn²⁺-Phos-tag (1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) attached with a 7-amino-4-methylcoumarin-3-acetic acid (AMCA). Carboxyfluorescein (FAM)-labeled phospho- and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, λ_em 520 nm) and AMCA (a fluorescent donor, λ_ex 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn²⁺-Phos-tag specifically captured the FAM-labeled phosphopeptide to form a stable 1:1 complex, resulting in efficient FRET. After the FAM-labeled phosphopeptide was dephosphorylated with alkaline phosphatase, the FRET disappeared. Using this FRET system, we demonstrated the detection of the time-dependent dephosphorylation of the FAM-labeled protein-tyrosine phosphatase 1B substrate.     

肝素对人类神经tau蛋白分子聚集及磷酸化的影响

在老年性痴呆患者的脑中,肝素与超磷酸化的tau蛋白共存[7].采用NCLK(neuronalcdc2-likekinase)及PP2B(phosphoproteinphosphatase2B)在含肝素的体系中对人类神经tau蛋白进行磷酸化和脱磷酸化,结果表明,肝素具有促进tau蛋白被磷酸化的功能,并促进该蛋白磷酸化分子二聚体的形成和单体的减少,其一级动力学常数分别为2.88×10-3s-1和1.74×10-3s-1.PP2B可使磷酸化的tau蛋白脱磷酸化,并且脱磷酸化作用随肝素浓度的增加而增强,提示肝素可能具有调节tau蛋白磷酸化状态的作用     

Protein-tyrosine Phosphatase 1B Antagonized Signaling by Insulin-like Growth Factor-1 Receptor and Kinase BRK/PTK6 in Ovarian Cancer Cells

Ovarian cancer, which is the leading cause of death from gynecological malignancies, is a heterogeneous disease known to be associated with disruption of multiple signaling pathways. Nevertheless, little is known regarding the role of protein phosphatases in the signaling events that underlie the disease; such knowledge will be essential to gain a complete understanding of the etiology of the disease and how to treat it. We have demonstrated that protein-tyrosine phosphatase 1B (PTP1B) was underexpressed in a panel of ovarian carcinoma-derived cell lines, compared with immortalized human ovarian surface epithelial cell lines. Stable restoration of PTP1B in those cancer cell lines substantially decreased cell migration and invasion, as well as proliferation and anchorage-independent survival. Mechanistically, the pro-survival IGF-1R signaling pathway was attenuated upon ectopic expression of PTP1B. This was due to dephosphorylation by PTP1B of IGF-1R β-subunit and BRK/PTK6, an SRC-like protein-tyrosine kinase that physically and functionally interacts with the IGF-1R β-subunit. Restoration of PTP1B expression led to enhanced activation of BAD, one of the major pro-death members of the BCL-2 family, which triggered cell death through apoptosis. Conversely, inhibition of PTP1B with a small molecular inhibitor, MSI-1436, increased proliferation and migration of immortalized HOSE cell lines. These data reveal an important role for PTP1B as a negative regulator of BRK and IGF-1Rβ signaling in ovarian cancer cells.