Ras-mediated cell proliferation and cell death: some clues from the interleukin-2 receptor system

Oncoproteins of the Ras family have been extensively studied because of their implication in human cancer. Their roles have been primarily assigned to the commandment of cell proliferation and suppression of apoptosis, which has also been demonstrated by the involvement of Ras activation in the signal transduction pathways triggered by most cytokine receptors. Nevertheless, the functions of Ras proteins have been extended in the last years by the findings showing that they can also act as promoters or enhancers of apoptosis in various systems and conditions. These considerations have raised the issue as to how the signals delivered by Ras are regulated and translated in terms of cellular responses, suggesting that signal complementation may direct the final fate of cells. As an example, the interleukin-2 receptor system may represent a useful model in which the meaning of Ras signals may be evaluated in terms of interactions with other simultaneous signalling events, since knowledge of the biochemical events triggered by the interaction of interleukin-2 with its cell surface receptor in lymphocytes has allowed the proposal of a complete signalling model arranged in three independent channels, one of which is mediated by Ras.This work was supported by grants from CICYT and Pharmacia-Upjohn.     

Taxonomy and function of C1 protein kinase C homology domains.

C1 domains are compact alpha/beta structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain 2 C1 domains (C1A and C1B) in their N-terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain-containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site-directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty-six C1 domains fit the profile for phorbol-ester binding and are termed "typical." Twenty-eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed "atypical." Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

低频脉冲电场对PC12细胞酪氨酸羟化酶和多巴胺合成的影响

运用Western印迹和HPLC分别测定不同时间电场刺激和刺激后不同培养时间条件下,PC12细胞内酪氨酸羟化酶(TH)和细胞培养液中多巴胺(DA)含量的变化。结果显示,受到短时间(5、10min)脉冲电场刺激的PC12细胞,经较短时间(2天)的培养后,细胞内TH的含量和培养液中DA的含量均比对照组有所提高,但随着培养时间的延长(3~5天),TH和DA的含量均明显下降。然而,长时间(15、20、30min)脉冲电场刺激组则先表现为TH和DA的合成受到抑制,但随着培养时间的延长,其合成则被逐渐激活。采用蛋白激酶A(PKA)特异性抑制剂H-89和有丝分裂原活化蛋白激酶的激酶(MEK1/2)特异性抑制剂U0126,研究脉冲电场刺激所激活的与TH和DA合成相关的信号通路。结果表明,在没有神经生长因子(NGF)存在的情况下,PC12细胞主要通过PKA通路来激活TH的合成,低频脉冲电场刺激也主要激活PKA通路,因为抑制这条信号通路能显著抑制电场刺激所诱导的TH合成。     

Sensitivity and specificity amplification in signal transduction

Intracellular signal transduction pathways transmit signals from the cell surface to various intracellular destinations, such as cytoskeleton and nucleus through a cascade of protein-protein interactions and activation events, leading to phenotypic changes such as cell proliferation, differentiation, and death. Over the past two decades, numerous signaling proteins and signal transduction pathways have been discovered and characterized. There are two major classes of signaling proteins: phosphoproteins (e.g., mitogen-activated protein kinases) and guanosine triphosphatases (GTPases; e.g., Ras and G proteins). They both function as molecular switches by addition and removal of one or more high-energy phosphate groups. This review discusses developments that seek to quantify the signal transduction processes with kinetic analysis and mathematical modeling of the signaling phosphoproteins and GTPases. These studies have provided insights into the sensitivity and specificity amplification of biological signals in integrated systems.     

Interactions of opioid and chemokine receptors: oligomerization of mu,kappa, and delta with CCR5 on immune cells

In this study, we examined the signaling pathways for extracellular signal-related protein kinase (ERK) activation by three structurally different peroxisome proliferator activated receptor-gamma (PPARgamma) agonists. In murine C2C12 myoblasts, treatment with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and GW1929 leads to ERK1/2 phosphorylation in a time- and concentration-dependent manner. Consistent with ERK phosphorylation, mitogen activated protein/ERK kinase (MEK) phosphorylation as well as Raf-1 kinase activity are also accordingly stimulated, while the constitutive Ser259 phosphorylation of Raf-1 is decreased. The ERK phosphorylation induced by PPARgamma agonists is not blocked by the PKC inhibitors GF109203X and Ro31-8220, the PI3K inhibitor wortmannin, the Ras inhibitor FPTI, the negative mutant of Ras, or the PPARgamma antagonist bisphenol A diglycidil ether. Expression of PPARgamma2 without DNA binding domain or with a nonphosphorylatable mutant (S112A) fails to change ERK phosphorylation by 15d-PGJ(2). On the contrary, the ERK phosphorylation by PPARgamma agonists is inhibited by the MEK inhibitor PD98059, GSH, and permeable SOD mimetic MnTBAP. Chemiluminescence study reveals that these three PPARgamma agonists are able to induce superoxide anion production, with an efficacy similar to their action on ERK phosphorylation. Consistent with this notion, we also show that superoxide anion donor 2,3-dimethoxy-1,4-naphoquinone elicits ERK phosphorylation. In this study, we for the first time demonstrate a novel mechanism, independent of Ras activation but initiated by superoxide anion production, for PPARgamma agonists to trigger the Raf-MEK-ERK1/2 signaling pathway.     

甲羟戊酸通路对细胞生长调控作用的研究进展

甲羟戊酸(MVA)通路对细胞生长具有重要的调节作用,MVA及其衍生物通过对蛋白质异戊烯化和N糖基化修饰而影响Ras蛋白、生长因子及受体的功能、细胞内信号转导和细胞的生长。MVA通路参与血管活性物质生成的调节是其调节细胞生长的另一机制。MVA生成的限速酶羟甲基戊二酸单酰辅酶A(HMGCoA)则受MVA通路衍生物的反馈抑制。HMGCoA还原酶抑制剂通过抑制MVA及其衍生物的生成而抑制细胞的生长和增殖。     

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src^Y527 and Ras^V12 activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.     

Ras guanyl nucleotide releasing protein 2 affects cell viability and cell–matrix adhesion in ECV304 endothelial cells

Ras guanyl nucleotide releasing proteins (RasGRPs) are guanine nucleotide exchange factors that activate Ras and Rap. We recently reported that xrasgrp2, which is a homolog of the human rasgrp2, plays a role in vasculogenesis and/or angiogenesis during early development of Xenopus embryos. However, the function of RasGRP2 in human vascular endothelium remains unknown. Therefore we aimed to analyze the function of human RasGRP2 in vascular endothelial cells. RasGRP2 overexpression did not increase Ras activation. However, it slightly increased Ras expression and increased proliferation in ECV304 cells. Furthermore, RasGRP2 overexpression increased Rap1 activation and cell–matrix adhesion in ECV304 cells. These data demonstrate that RasGRP2 increases cell viability and cell–matrix adhesion through increased Ras expression and Rap1 activation, respectively, in endothelial cells.