蛋白激酶对活性氧调节7721人肝癌细胞生长的影响

以有义和反义人锰型超氧化物歧化酶 (MnSOD)、蛋白激酶B (PKB)cDNA分别转染人 772 1肝癌细胞系建立高、低表达MnSOD和PKB的模型 ,通过改变细胞内、外源性活性氧和抗氧化水平及PKB的活性 ,研究活性氧调节肝癌细胞生长过程中与PKB信号转导途径的关系。结果表明 ,低浓度外源性活性氧过氧化氢 (1～ 10μmol/L)应激及MnSOD低表达细胞中内源性活性氧水平升高 ,都可促进肝癌细胞增殖 ,而抗氧化剂丹参素 (4 0mg/L)干预及MnSOD高表达使细胞内源性活性氧水平相对下降 ,都可一定程度抑制细胞的生长。采用3 2 P参入法检测细胞PKB酶活性 ,发现细胞内、外源性活性氧均能激活PKB ,而采用抗氧化干预 ,能明显抑制PKB的酶活性。采用RT PCR法检测AP 1转录因子组成成员c fos和c jun基因的mRNA表达 ,发现与PKB活性呈正比。说明活性氧在特定细胞内氧化 /还原环境下可通过激活PKB途径传递信号、调控转录因子AP 1表达、促进肝癌细胞生长     

蛋白激酶B对活性氧调节7721人肝癌细胞生长的影响

以有义和反义人猛型超氧化物歧化酶（MnSOD）、蛋白激酶B（PKB）cDNA分别转染入7721肝癌细胞系建立高、低表达MnSOD和PKB的模型,通过改变细胞内、外源性活性氧和抗氧水平及PKB的活性,研究活性氧调节肝癌细胞生长过程中与PKB信号转导途径的关系。结果表明,低浓度外源性活性氧过氧化氢（1～10umol/L）应激及MnSOD低表达细胞中内源性活性氧水平升高,都可促进肝癌细胞增殖,而抗氧化剂丹参素(40mg/L）干预及MnSOD高表达使细胞内源性活性氧水平相对下降,都可一定程度抑制细胞的生长。采用^32P参入法检测细胞PKB酶活性,发现细胞内、外源性活性氧均能激活PKB,而采用抗氧化干预,能明显抑制PKB的酶活性。采用PT-PCR法检测AP-1转录因子组成成员c-fos和c-jun基因的mRNA表达,发现与PKB活性呈正比。说明活性氧在特定细胞内氧化/还原环境下可通过激活PKB途径传递信号、调控转录因子AP-1表达、促进肝癌细胞生长。     

受体蛋白激酶和植物发育

受体蛋白激酶是蛋白激酶家族中的重要一类。根据其胞外受体结构域的组成不同 ,植物受体蛋白激酶可划分为不同的类型。近些年的研究发现 ,蛋白激酶是植物发育和抗性反应中重要成分 ,是信号分子的重要受体 ,在信号传导过程中起着重要作用。随着对植物发育过程中信号传导机理认识的不断深入 ,人们有望通过操作植物发育过程向人们需要的方向发展 ,达到控制果实的大小和提高产量的目的。     

PKB/Akt:一个具有多种功能的蛋白激酶

蛋白激酶B(protein kinaseB,PKB)是细胞信号传导过程中的一个重要的中间体。在过去lO多年的研究中,发现它不仅参与调节细胞糖代谢、细胞增殖、细胞凋亡,而且与糖尿病和癌症的发生也有关。目前PKB的相关调控机制还未得到完全阐明。在此,作者谨就PKB的一些研究进展作一介绍。     

蛋白激酶B(PKB/Akt)的结构、调控与功能

原癌基因编码的蛋白质丝/苏氨酸激酶PKB/Akt在细胞代谢、细胞存活、细胞周期调控、转录调控等多种生物学过程中发挥着重要作用。该文综述了PKB/Akt的结构、调控机制及功能。     

钙依赖型蛋白激酶调节保卫细胞膜内向钾离子通道的实验证据

保卫细胞内钙离子浓度的变化对气孔保卫细胞质膜上的内向钾离子通道活性有显著调节作用,而钾离子通道活性的变化可导致细胞渗透压的改变,进而调节气孔的开闭运动。然而,钙离子通过何种机制实现对钾离子通道的调节尚不清楚。作者利用膜片钳全细胞记录方法探讨了钙依赖型蛋白激酶（ＣＤＰＫ）是否参与了钙离子调节保卫细胞钾离子通道的信号转导过程。当胞内钙离子浓度为１．５μｍｏｌ／Ｌ时,保卫细胞全细胞内向钾电流被抑制约６０％;同时加入ＣＤＰＫ的底物组蛋白ⅢＳ或ＣＤＰＫ的底物竞争性抑制剂鱼精蛋白可完全逆转钙离子的作用;但在胞内同时加入纯化的ＣＤＰＫ蛋白则可进一步促进钙离子对保卫细胞内向钾电流的抑制。研究结果初步证明,ＣＤＰＫ介导了钙离子调节保卫细胞内向钾离子通道的信号转导过程     

蛋白激酶的研究 (综述)

近年来,蛋白激酶研究进展较快,本文综述蛋白激酶的种类、结构、细胞定位,讨论几种植物蛋白激酶及其与信号转导的关系。     

植物受体蛋白激酶的研究进展

在植物中存在一种由胞外结构域、跨膜区域和胞内的蛋白激酶区域三部分组成的跨膜受体蛋白激酶（receptor-lik protein kinases,RLKs)。该蛋白一方面作为胞外特异配基的受体,同时本身又是一种蛋白激酶。研究表明,植物细胞中的RLKs可能参与了植物细胞抗逆反应,植物形态发生、自交不亲和等生理生化反应,作者将从RLKs的结构、种类,基因表达方式及其植物生长和发育过程中的作用做简要介绍。     

血清和糖皮质激素调节蛋白激酶2α过表达通过Wnt/β-Catenin信号通路抑制肝癌细胞株BEL7402增殖

血清和糖皮质激素调节蛋白激酶(SGK)家族参与调节生长因子和激素的信号转导.为了研究SGK家族成员SGK2α在细胞中的功能,构建了真核表达质粒pEGFP-N1-SGK2α并瞬时转染HEK293细胞,通过激光共聚焦显微镜观察发现融合蛋白SGK2α-GFP主要定位于细胞浆,免疫共沉淀实验发现SGK2α与糖原合成激酶3β(GSK3β)存在相互作用.利用PCDNA6-V5-HisB-SGK2α质粒转染肝癌BEL7402细胞,建立稳定表达SGK2α蛋白的细胞系,通过细胞增殖实验发现,SGK2α的过表达使BEL7402细胞生长速度减慢、细胞倍增时间延长.裸鼠成瘤实验发现,与对照组细胞相比表达SGK2α的BEL7402细胞在裸鼠中的成瘤能力明显降低.免疫印迹实验证实,SGK2α的过表达不影响GSK3β的表达,但却使β-catenin和Cyclin D1的表达下调.提示影响Wnt/β-catenin信号通路关键分子的表达可能是外源性SGK2α蛋白过表达抑制BEL7402细胞增殖的分子机制.     

蛋白激酶C对酪氨酸蛋白激酶系统的调节

本文论述了蛋白激酶Ｃ（ＰＫＣ）对生长因子受体激活的Ｒａｓ／ＭＡＰＫ途径的多种调节,其中,有直接的也有间接的,有激活也有抑制。这些不同水平不同性质的调节使细胞对外来刺激作出相应的反应。由于ＰＫＣ和Ｃａ＾２＋是肌醇磷脂系统中十分重要的两个组分,本文所论述的也是目前所知的肌醇磷脂系统对酪氨酸蛋白激酶系统调节的主要过程。     

Serum and glucocorticoid-regulated protein kinases: variations on a theme

The phosphatidylinositol 3' kinase (PI3K)-signaling pathway plays a critical role in a variety of cellular responses such as modulation of cell survival, glucose homeostasis, cell division, and cell growth. PI3K generates important lipid second messengers-phosphatidylinositides that are phosphorylated at the 3' position of their inositol ring head-group. These membrane restricted lipids act by binding with high affinity to specific protein domains such as the pleckstrin homology (PH) domain. Effectors of PI3K include molecules that harbor such domains such as phosphoinositide-dependent kinase (PDK1) and protein kinase B (PKB), also termed Akt. The mammalian genome encodes three different PKB genes (alpha, beta, and gamma; Akt1, 2, and 3, respectively) and each is an attractive target for therapeutic intervention in diseases such as glioblastoma and breast cancer. A second family of three protein kinases, termed serum and glucocorticoid-regulated protein kinases (SGKs), is structurally related to the PKB family including regulation by PI3K but lack a PH domain. However, in addition to PH domains, a second class of 3' phosphorylated inositol phospholipid-binding domains exists that is termed Phox homology (PX) domain: this domain is found in one of the SGKs (SGK3). Here, we summarize knowledge of the three SGK isoforms and compare and contrast them to PKB with respect to their possible importance in cellular regulation and potential as therapeutic targets.     

Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3+ Regulatory T Cells

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Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.     

粘着斑激酶在TNF-α作用下影响SMMC-7721细胞PKB的表达水平

 探讨在肿瘤坏死因子α(TNF-α)作用下 ,粘着斑激酶 (focal adhesion kinase,FAK)对蛋白激酶 B(PKB)蛋白水平的影响 .利用构建、转染 FAK反义质粒来特异性降低 SMMC- 772 1细胞的FAK含量 ,及用 Western杂交的方法来检测 PKB的蛋白含量 .文献报道 TNF- α能够激活磷脂酰肌醇 3-激酶 (PI3K)而使 PKB发生磷酸化 .但是至于 TNF-α对 PKB蛋白水平的影响目前并无报道 .研究发现 ,当用 wortmannin特异性抑制 PI3K活性后可以显著降低 PKB的蛋白含量 .提示PI3K对维持 PKB的基础蛋白水平是必需的 .但是 TNF- α本身对 PKB的蛋白水平无明显影响 .而当用不同浓度的 TNF- α和 wortmannin处理 SMMC- 772 1细胞时 ,发现 PKB的蛋白含量随着TNF-α浓度的增加而降低 .提示 TNF-α可能除了通过 PI3K外 ,还可能通过另一条途径来下调PKB的表达 .而当用 FAK反义质粒转染 SMMC- 772 1细胞后 (FAK下降了 60 % ) ,发现在当用不同浓度的 TNF- α处理的情况下 ,FAK反义质粒转染株 AS- 772 1细胞的 PKB含量降低为对照的70 % ;而在用 TNF-α和 wortmannin处理的情况下 ,下降为对照的 40 %～ 60 % .TNF-α能够通过PI3K及另一未知途径来影响 PKB的蛋白水平 .而 FAK在 TNF- α作用下能够不通过 PI3K来影响PKB的蛋白水平 .     

Retinoid X Receptor-selective Signaling in the Regulation of Akt/Protein Kinase B Isoform-specific Expression

The differentiation and fusion of myoblasts into mature myotubes are complex processes responding to multiple signaling pathways. The function of Akt/PKB is critical for myogenesis, but less is clear as to the regulation of its isoform-specific expression. Bexarotene is a drug already used clinically to treat cancer, and it has the ability to enhance the commitment of embryonic stem cells into skeletal muscle lineage. Whereas bexarotene regulates fundamental biological processes through retinoid X receptor (RXR)-mediated gene expression, molecular pathways underlying its positive effects on myogenesis remain unclear. In this study, we have examined the signaling pathways that transmit bexarotene action in the context of myoblast differentiation. We show that bexarotene promotes myoblast differentiation and fusion through the activation of RXR and the regulation of Akt/PKB isoform-specific expression. Interestingly, bexarotene signaling appears to correlate with residue-specific histone acetylation and is able to counteract the detrimental effects of cachectic factors on myogenic differentiation. We also signify an isoform-specific role for Akt/PKB in RXR-selective signaling to promote and to retain myoblast differentiation. Taken together, our findings establish the viability of applying bexarotene in the prevention and treatment of muscle-wasting disorders, particularly given the lack of drugs that promote myogenic differentiation available for potential clinical applications. Furthermore, the model of bexarotene-enhanced myogenic differentiation will provide an important avenue to identify additional genetic targets and specific molecular interactions that we can study and apply for the development of potential therapeutics in muscle regeneration and repair.     

Loss of Serum and Glucocorticoid-Regulated Kinase 3 (SGK3) Does Not Affect Proliferation and Survival of Multiple Myeloma Cell Lines

Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells—either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.     

Glutamate Activates Protein Kinase B (PKB/Akt) through AMPA Receptors in Cultured Bergmann Glia Cells

Glutamate is involved in gene expression regulation in neurons and glial cells through the activation of a diverse array of signaling cascades. In Bergmann glia, Ca²⁺-permeable α-hydroxy-5-methyl-4-isoazole-propionic acid (AMPA) receptors become tyrosine phosphorylated after ligand binding and by these means form multiprotein signaling complexes. Of the various proteins that associate to these receptors, the phosphatidylinositol 3-kinase (PI-3K) deserves special attention since D3-phosphorylated phosphoinositides are docking molecules for signaling proteins with a pleckstrin homology domain. In order to characterize the role of PI-3K in AMPA receptors signaling, in the present report we analyze the involvement of the serine/threonine protein kinase B in this process. Our results demonstrate an augmentation in protein kinase B phosphorylation and activity after glutamate exposure. Interestingly, the effect is independent of Ca²⁺ influx, but sensitive to Src blockers. Our present findings broaden our current knowledge of glial glutamate receptors signaling and their involvement glutamatergic neurotransmission.Special issue dedicated to Miklós Palkovits.