Oxidants in receptor tyrosine kinase signal transduction pathways

The accumulation of oxygen in the atmosphere created an evolutionary stress for organisms to survive because oxygen, while the by-product of photosynthesis and an important substrate in oxidative metabolism, can also be partially reduced to form toxic products. These forms of oxygen, reduced by one electron or two electrons, yield superoxide anion (O(2).-) and hydrogen peroxide (H(2)O(2)), respectively. Recent studies suggest that reactive oxygen species (ROS) such as O(2).- and H(2)O(2) function as mitogenic mediators of activated growth-factor receptor signaling. Reported data imply that growth factor-stimulated ROS generation can mediate intracellular signaling pathways by activating protein tyrosine kinases, inhibiting protein tyrosine phosphatase, and regulating redox-sensitive gene expression. This review examines the mechanisms of growth factor-induced generation of ROS and their roles in specific receptor tyrosine kinase signaling pathways.     

Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation

The molecular details of mammalian stress-activated signal transduction pathways have only begun to be dissected. This, despite the fact that the impact of these pathways on the pathology of chronic inflammation, heart disease, stroke, the debilitating effects of diabetes mellitus, and the side effects of cancer therapy, not to mention embryonic development, innate and acquired immunity, is profound. Cardiovascular disease and diabetes alone represent the most significant health care problems in the developed world. Thus it is not surprising that understanding these pathways has attracted wide interest, and in the past 10 years, dramatic progress has been made. Accordingly, it is now becoming possible to envisage the transition of these findings to the development of novel treatment strategies. This review focuses on the biochemical components and regulation of mammalian stress-regulated mitogen-activated protein kinase (MAPK) pathways. The nuclear factor-kappa B pathway, a second stress signaling paradigm, has been the subject of several excellent recent reviews (258, 260).     

GPER信号转导通路

G蛋白偶联雌激素受体(GPER)是上世纪90年代发现的一种新型的雌激素受体,它可以独立于经典的雌激素核受体介导雌激素的多种生物学功能。GPER可通过激活EGFR和c AMP介导雌激素的快速非基因组效应,GPER还可与其它多个信号转导通路进行对话。因此,本文就GPER信号转导通路作以综述。     

酪氨酸蛋白激酶JAK1 在IL-6诱导JAK/STAT 途径活化中的作用

目的 酪氨酸蛋白激酶JAK1和转录因子STAT3为参与IL－6诱导的JAK／STAT信号转导途径的两种主要的信号蛋白分子。本研究试图揭示JAK1在JAK／STAT途径诱导活化中的作用。方法 分别采用凝胶阻滞电泳（EMSA）和免疫沉淀（IP）法观察IL－6刺激作用下STAT3和JAK1在3种骨髓瘤细胞系（XG－7，KM－3和Sko-007)中的诱导活化状态。采用RTPCR和Western-blot法检测这两种信号蛋白分子在以上3株靶细胞中的表达情况。结果 尽管SAT3在3株靶细胞中都能够正常表达，但只有Sko-007细胞中出现IL－6刺激作用下STAT3的诱导活化。在XG－7细胞中，既没有检测到JAK1的表达，也没有观察到JAK1的活化。尽管JAK1在KM－3细胞中能够正常表达，但不能被IL－6诱导激活。Sko-007细胞中则同时出现JAK1的表达及IL－6刺激后的诱导活化。结论 JAK1的正常表达和激活是IL－6刺激作用下JAK／STAT信号转导途径在骨髓瘤细胞中诱导活化的前提条件。     

Rapid electrical stimulation induces early activation of kinase signal transduction pathways and apoptosis in adult rat ventricular myocytes

Chronic tachycardia in patients and rapid pacing in animal models induce myocardial dysfunction and initiate a cascade of compensatory adaptations that are ultimately unsustainable, leading to ventricular enlargement and failure. The molecular pathogenesis during the early stages of tachycardia-induced cardiomyopathy, however, remains unclear. We utilized our previously reported cell culture pacing system to directly assess phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signalling of adult rat ventricular myocytes (ARVM) in response to rapid electrical stimulation. Freshly isolated ARVMs were maintained quiescent (0 Hz), or continuously stimulated at 5 (normofrequency) and 8 Hz (rapid frequency). Pacing resulted in an increase in mitochondrial respiration, assessed by mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 48 h. Rapid pacing at 8 Hz significantly increased cell injury and death as assessed by Trypan Blue uptake, creatine phosphokinase release, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Pacing at 5 Hz induced early, but weak, activation of Akt and protein kinase 38 (p38). Rapid pacing further augmented the early activation of Akt and p38, and induced extracellular signal-related kinase (Erk) and c-jun amino terminal kinase (JNK) activation. Incubation of ARVM with PI3K inhibitor LY294002 resulted in a twofold increase of TUNEL-positive cells under all pacing conditions examined. In conclusion, rapid pacing has immediate and detrimental consequences for cardiomyocyte survival, with pro-apoptotic pathways (e.g. JNK, p38) able to overwhelm antiapoptotic signalling (PI3K/Akt, Erk). The rapid pacing methodology described in this report will be particularly useful in determination of cell signalling pathways associated with tachycardia-induced cardiomyopathy.     

Activation of the Syk tyrosine kinase is insufficient for downstream signal transduction in B lymphocytes

Background  

Immature B lymphocytes and certain B cell lymphomas undergo apoptotic cell death following activation of the B cell antigen receptor (BCR) signal transduction pathway. Several biochemical changes occur in response to BCR engagement, including activation of the Syk tyrosine kinase. Although Syk activation appears to be necessary for some downstream biochemical and cellular responses, the signaling events that precede Syk activation remain ill defined. In addition, the requirements for complete activation of the Syk-dependent signaling step remain to be elucidated.     

Signal transduction pathways for activation of extracellular signal-regulated kinase by arachidonic acid in rat neutrophils

Phosphorylation of extracellular signal-regulated kinase (ERK) in response to arachidonic acid (AA) was rapid and transient, peaking at 1 min and disappearing after 3 min, and it was accompanied by an increase in ERK activity in rat neutrophils. We examined the upstream regulation of AA-stimulated ERK activation using one of the following signaling pathway inhibitors to pretreat rat cells: the ERK kinase inhibitor U0126 or PD98059, the G(i/o) inhibitor pertussis toxin (PTX), the tyrosine kinase inhibitor genistein, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or LY294002, the Ca2+ chelator 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, or the phospholipase C (PLC) inhibitor U73122. All of these inhibitors attenuated AA-induced ERK activation. Activation of ERK was also effectively attenuated by the cyclooxygenase and lipoxygenase inhibitor BW755C and by the leukotriene biosynthesis inhibitor MK886, but the cyclooxygenase inhibitor indomethacin did not attenuate ERK activation. After exposing cells to three distinct protein kinase C (PKC) inhibitors, we found that G?6976 significantly attenuated ERK phosphorylation but potentiated ERK activity. Neither G?6983 nor GF109203X affected AA-induced responses. These data suggest that the lipoxygenase metabolite(s) produced mediates AA-stimulated ERK activation and that this effect is upstream regulated by PT-sensitive G protein, non-receptor tyrosine kinase, PI3K, and PLC/Ca2+ signaling pathways in rat neutrophils.     

Interleukin-2 signal transduction in human NK cells: multisite phosphorylation and activation of the tyrosine kinase p56lck.

Interleukin-2 (IL-2) potently stimulates natural killer (NK) cell proliferation and cytotoxic function. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the electrophoretic mobility of p56lck. The present studies indicate that IL-2 induces a rapid (< or = 1 min) increase in the catalytic activity of p56lck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56lck itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorylation of the NH2-terminal region of p56lck containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p56lck. These results suggest that p56lck serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56lck.     

Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events

Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.     

Immunohistochemical screening for oncogenic tyrosine kinase activation

Tyrosine kinases causing the abnormal phosphorylation of intracellular proteins have been shown to contribute to oncogenic transformation in a number of human neoplasms. Immunohistological staining of routine biopsy sections for increased levels of phosphotyrosine may therefore provide a simple means of screening for tumours containing activated tyrosine kinases. In this study, monoclonal antibodies to phosphotyrosine were used to immunostain a cell line and tumour biopsies from lymphomas known to contain the activated anaplastic-lymphoma-kinase (ALK) tyrosine kinase. A range of normal and other neoplastic tissues were also immunostained for comparison. An anaplastic large cell lymphoma (ALCL) cell line carrying the (2;5) translocation, which creates the activated nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) tyrosine kinase, was strongly labelled. Routine tissue biopsies from five cases of ALK-positive ALCL were also strongly positive for phosphotyrosine. The characteristic granular cytoplasmic labelling pattern for phosphotyrosine observed in a B-cell lymphoma (expressing full length ALK kinase) was identical to that obtained using an ALK-specific antibody, thus confirming that labelling for phosphotyrosine in lymphoma cells reflects the presence of an activated kinase. When normal lymphoid tissues were stained, there was little or no labelling for phosphotyrosine, but stronger labelling was seen in other cells and tissues; for example, endothelial cells and some carcinoma samples. Whilst the strong labelling for phosphotyrosine observed in the lymphoma cells is due to the presence of activated ALK, the strong staining of some normal cells presumably represents physiologically active kinases and this should be taken into account when interpreting the immunostaining of non-lymphoid tumours. The simplicity of this method, however, means that it offers a new rapid approach to the screening of large numbers of tumours for the presence of aberrant tyrosine kinase activation, particularly if they arise from tissues which normally contain only background levels of phosphotyrosine. Copyright © 1999 John Wiley & Sons, Ltd.     

Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation.

Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation.     

抗CD28＋B7.1单抗共刺激PBLs诱导肝癌细胞凋亡的蛋白激酶信号转导

目的 研究Ｔ淋巴细胞活化、增殖、介导肝癌细胞凋亡过程中其蛋白激酶Ｃ（ＰＫＣ）和酪氨酸蛋白激酶（ＴＰＫ）的活性变化。方法 用抗ＣＤ２８＋Ｂ７．１（ＣＤ８０）单克隆抗体共刺激正常人外周血淋巴细胞（ＰＢＬｓ）后作用于肝癌细胞（ＢＥＬ－７４０２）。结果 激活的Ｔ细胞中ＰＫＣ、ＴＰＫ活性明显增强,并与肝癌细胞凋亡程度呈正相关。结论 ＰＫＣ、ＴＰＫ在Ｔ细胞活化、增殖及诱导肝癌细胞凋亡的信号转导中起重要作用。     

丝裂原活化蛋白激酶信号转导通路与脓毒症研究进展

近年来,关于脓毒症的发生机制,尤其是细胞内信号转导机制备受人们关注.目前研究表明,丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)可能在介导脓毒症时大量炎性因子的释放中起着重要作用.本文就MAPK信号转导途径在脓毒症过程中的活化、对炎症因子的调控作用以及MAPK信号通路抑制剂在脓毒症中的应用作一综述.     

Alterations in tyrosine protein kinase activities upon activation of human neutrophils

Human neutrophils are phagocytic cells that can be activated by a variety of agonists to undergo a group of physiologic responses. This "stimulus-response" coupling is thought to be dependent on the phosphorylation of specific proteins. We have previously shown that, in addition to the widely distributed serine and threonine protein kinases, neutrophils contain tryosine protein kinase activity in the cell cytosol and particulate fractions. When neutrophils are treated with a variety of agents, phosphorylation of both cytosolic and particulate proteins on tyrosine residues occurs. Increases in tyrosine phosphorylation may be a result of increases in the activity of tyrosine kinases or a decrease in the activity of phosphotyrosine phosphatases. In this study, we have used a novel nondenaturing polyacrylamide gel electrophoretic method to demonstrate that treatment of human neutrophils with the chemotactic peptide FMLP or the phorbol ester phorbol myristate acetate induces a time- and concentration-dependent increase in the tyrosine protein kinase activity found in the cell cytosol and cell particulate fraction. The time course and concentration range over which these changes occur are similar to those seen for activation of the neutrophil oxidative burst and phosphorylation of proteins on tyrosine residues, suggesting that these events may be related.     

Protein kinase mediators of integrin signal transduction

 Protein kinases are important mediators of signal transduction initiated by soluble growth factors and cytokines. Cellular interactions with the extracellular matrix are mediated largely by members of the integrin class of cell adhesion molecules, which also subsume signal transduction functions required for cell growth, differentiation, and survival. Here we review the involvement of protein kinases in mediating integrin intracellular signal transduction and the possible role for these molecules in regulating integrin adhesion. Although in most cases mechanistic details are incomplete, the emerging theme of protein kinases mediating cross-talk between growth factor receptor and integrin signalling systems provides a timely backdrop against which to present new developments in this area. The contribution of the actin cytoskeleton to integrin signal transduction is discussed, with respect to the concept of ’solid-state’ signalling providing a mechanism for imposing order on the protein-protein interactions which underlie signal discrimination. Moreover, we review evidence that dysregulated integrin signalling contributes to pathological processes including arthritis, thrombasthenia, leucocyte adhesion deficiencies, and tumour angiogenesis and invasion. Received: 14 May 1996 / Accepted: 2 July 1996     

STE11 is a protein kinase required for cell-type-specific transcription and signal transduction in yeast

The STE11 gene of Saccharomyces cerevisiae is one of several genes required for mating between two haploid cell types of this yeast. Its product is required for response to a signal that causes arrest of the mitotic cell cycle in the G1 phase and induction of mating-type-specific genes. The nucleotide sequence of the STE11 gene was determined. The predicted amino acid sequence shows homology to the protein kinase family. We demonstrate that the STE11 product has kinase catalytic activity and that this activity is required for its in vivo functions.