BACKGROUND Ras are small cellular GTPases which regulate diverse cellular processes. It has three isoforms: H-Ras, K-Ras, and N-Ras. Owing to the N-terminus (1-165 residues) sequence homology these isoforms were thought to be functionally redundant. However, only K-Ras-deficient mice but not H-Ras- and N-Ras-deficient mice show embryonic lethality. Similarly, mutations in a given Ras isoform are associated with a particular type of cancer. Moreover, we have previously reported that Ras isoforms perform unique functions in Leishmania major infection. Thus, Ras isoforms are implicated to have signaling and functional specificity but the mechanism remains to be elucidated. RESULT Using CD40 as a model receptor, we showed that depending on the strength of signaling, specific Ras isoforms are activated. Weak CD40 signal activates N-Ras, whereas strong signal activates H-Ras and K-Ras. Additionally, we showed that suppression of N-Ras expression reduced CD40-induced extracellular signal-regulated kinase-1/2 (ERK-1/2) activation and Interleukin (IL)-10 production; whereas suppression of H-Ras or K-Ras reduced CD40-induced p38 mitogen-activated protein kinase (p38MAPK) activation and IL-12 production. Furthermore, we showed that Ras isoforms have activator (GEF) specificity as weak CD40 signal-activated N-Ras requires Sos-1/2 whereas strong CD40 signal-activated H-Ras/K-Ras requires Ras-GRP as the guanine-nucleotide exchange factor (GEF) inducing ERK-1/2- or p38MAPK-mediated IL-10 or IL-12 productions, respectively, in macrophages. Silencing of syk reduced CD40-induced N-Ras activation but silencing of lyn inhibited H-Ras and K-Ras activation. In CD40 signaling, Ras isoforms also showed effector specificity; while H-Ras and K-Ras showed specificity for phosphatidyl inositol-3 kinase activation at high dose of CD40 stimulation, N-Ras primarily associated with Raf-1 at low dose of CD40 stimulation. Moreover, fractal analysis showed that functional site surface roughness for H-Ras (SurfaceFD = 2.39) and K-Ras (SurfaceFD = 2.39) are similar but significantly different from N-Ras (SurfaceFD = 2.25). CONCLUSION The activator and effector specificities of Ras isoforms in CD40 signaling indicated their differential involvement in CD40 pathway and in maintaining the reciprocity. Our observations reveal Ras-regulated signaling outcome and its potential for developing Ras isoform-targeted immunotherapy and prophylaxis.

中文翻译：

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Ras亚型：CD40途径的信号特异性。

背景技术Ras是调节各种细胞过程的小型细胞GTP酶。它具有三种同工型：H-Ras，K-Ras和N-Ras。由于N末端（1-165个残基）的序列同源性，这些同工型被认为在功能上是多余的。但是，只有K-Ras缺陷小鼠而不是H-Ras和N-Ras缺陷小鼠表现出胚胎致死性。类似地，给定的Ras亚型的突变与特定类型的癌症有关。此外，我们以前曾报道过Ras亚型在利什曼原虫的严重感染中具有独特的功能。因此，暗示Ras同工型具有信号传导和功能特异性，但是其机理尚待阐明。结果使用CD40作为模型受体，我们显示了根据信号强度，特定的Ras亚型被激活。CD40信号弱会激活N-Ras，而强信号会激活H-Ras和K-Ras。此外，我们显示抑制N-Ras表达减少了CD40诱导的细胞外信号调节激酶-1/2（ERK-1 / 2）激活和白介素（IL）-10的产生；而抑制H-Ras或K-Ras可减少CD40诱导的p38丝裂原活化蛋白激酶（p38MAPK）活化和IL-12产生。此外，我们显示Ras同工型具有激活剂（GEF）特异性，因为弱CD40信号激活的N-Ras需要Sos-1 / 2，而强CD40信号激活的H-Ras / K-Ras需要Ras-GRP作为鸟嘌呤核苷酸交换因子（GEF）分别在巨噬细胞中诱导ERK-1 / 2或p38MAPK介导的IL-10或IL-12产生。syk沉默降低CD40诱导的N-Ras激活，但lyn沉默抑制H-Ras和K-Ras激活。在CD40信令中 Ras亚型也表现出效应子特异性。H-Ras和K-Ras在高剂量CD40刺激下显示出磷脂酰肌醇3激酶激活的特异性，而N-Ras在低剂量CD40刺激下主要与Raf-1相关。此外，分形分析表明，H-Ras（SurfaceFD = 2.39）和K-Ras（SurfaceFD = 2.39）的功能部位表面粗糙度相似，但与N-Ras（SurfaceFD = 2.25）明显不同。结论Ras亚型在CD40信号传导中的激活子和效应子特异性表明它们在CD40途径和维持互易性方面存在差异。我们的观察结果揭示了Ras调控的信号转导结果及其在开发以Ras亚型为靶标的免疫疗法和预防疾病方面的潜力。H-Ras和K-Ras在高剂量CD40刺激下显示出磷脂酰肌醇3激酶激活的特异性，而N-Ras在低剂量CD40刺激下主要与Raf-1相关。此外，分形分析表明，H-Ras（SurfaceFD = 2.39）和K-Ras（SurfaceFD = 2.39）的功能部位表面粗糙度相似，但与N-Ras（SurfaceFD = 2.25）明显不同。结论Ras亚型在CD40信号传导中的激活子和效应子特异性表明它们在CD40途径和维持互易性方面存在差异。我们的观察结果揭示了Ras调控的信号转导结果及其在开发以Ras亚型为靶标的免疫疗法和预防疾病方面的潜力。H-Ras和K-Ras在高剂量CD40刺激下显示出磷脂酰肌醇3激酶激活的特异性，而N-Ras在低剂量CD40刺激下主要与Raf-1相关。此外，分形分析表明，H-Ras（SurfaceFD = 2.39）和K-Ras（SurfaceFD = 2.39）的功能部位表面粗糙度相似，但与N-Ras（SurfaceFD = 2.25）明显不同。结论Ras亚型在CD40信号传导中的激活子和效应子特异性表明它们在CD40途径和维持互易性方面存在差异。我们的观察结果揭示了Ras调控的信号转导结果及其在开发以Ras亚型为靶标的免疫疗法和预防疾病方面的潜力。分形分析表明，H-Ras（SurfaceFD = 2.39）和K-Ras（SurfaceFD = 2.39）的功能部位表面粗糙度相似，但与N-Ras（SurfaceFD = 2.25）明显不同。结论Ras亚型在CD40信号传导中的激活子和效应子特异性表明它们在CD40途径和维持互易性方面存在差异。我们的观察结果揭示了Ras调控的信号转导结果及其在开发以Ras亚型为靶标的免疫疗法和预防疾病方面的潜力。分形分析表明，H-Ras（SurfaceFD = 2.39）和K-Ras（SurfaceFD = 2.39）的功能部位表面粗糙度相似，但与N-Ras（SurfaceFD = 2.25）明显不同。结论Ras亚型在CD40信号传导中的激活子和效应子特异性表明它们在CD40途径和维持互易性方面存在差异。我们的观察结果揭示了Ras调控的信号转导结果及其在开发以Ras亚型为靶标的免疫疗法和预防疾病方面的潜力。结论Ras亚型在CD40信号传导中的激活子和效应子特异性表明它们在CD40途径和维持互易性方面存在差异。我们的观察结果揭示了Ras调控的信号转导结果及其在开发以Ras亚型为靶标的免疫疗法和预防疾病方面的潜力。结论Ras亚型在CD40信号传导中的激活子和效应子特异性表明它们在CD40途径和维持互易性方面存在差异。我们的观察结果揭示了Ras调控的信号转导结果及其在开发以Ras亚型为靶标的免疫疗法和预防疾病方面的潜力。