Protein Farnesylation-Dependent Raf/Extracellular Signal-Related Kinase Signaling Links to Cytoskeletal Remodeling to Facilitate Glucose-Induced Insulin Secretion in Pancreatic β-Cells
Protein Farnesylation-Dependent Raf/Extracellular Signal-Related Kinase Signaling Links to Cytoskeletal Remodeling to Facilitate Glucose-Induced Insulin Secretion in Pancreatic β-Cells
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DOI：
10.2337/db09-1334
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发表时间：
2010-04-01
期刊：
影响因子：
7.7
通讯作者：
Koch, Brandon J.
中科院分区：
文献类型：
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作者：
Kowluru, Anjaneyulu;Veluthakal, Rajakrishnan;Koch, Brandon J.
目的：小G蛋白的翻译后预基化(如法尼化)被认为是细胞骨架重塑和分泌囊泡与质膜融合所必需的。在这里，我们研究了蛋白法尼化在参与Ral-1/细胞外信号相关激酶(ERK1/2)信号通路的信号步骤中的作用，该信号通路参与了葡萄糖诱导的Rac1激活和胰岛胰岛素分泌。研究设计与方法-这些研究在INS 832/13细胞和正常大鼠胰岛中进行。分子生物学(如过表达或小干扰RNA[siRNA]介导的基因敲除)和药理学方法被用来确定法尼化在葡萄糖介导的ERK1/2、rac1和胰岛素分泌激活中的作用。Western blotting检测ERK1/2的激活情况。P21激活的蛋白激酶下拉试验测定RACL的活性(即Rac1.GTP)。结果-提供结构特异性的法尼基转移酶抑制剂(FTI-277或FTI-2628)或siRNA介导的FTaseβ亚基下调可显著抑制葡萄糖刺激的ERK1/2和RACL激活和胰岛素分泌。用GW-5074药理抑制Raf-1激酶可显著降低葡萄糖对ERK1/2磷酸化、Rac1激活和胰岛素分泌的刺激作用，提示Raf-1激活可能位于ERK1/2和Rac1激活的上游，导致葡萄糖诱导的胰岛素释放。最后，siRNA介导的ERK1/2内源性表达沉默显著减弱了葡萄糖诱导的RacL激活和胰岛素分泌。结论：我们的研究结果首次证明蛋白质法尼化在葡萄糖介导的Raf/ERK信号通路中发挥作用，最终导致rac1的激活，这已被证明是细胞骨架重组和胰岛素分泌所必需的。糖尿病59：967-977,2010
OBJECTIVE-Posttranslational prenylation (e.g., farnesylation) of small G-proteins is felt to be requisite for cytoskeletal remodeling and fusion of secretory vesicles with the plasma membrane. Here, we investigated roles of protein farnesylation in the signaling steps involved in Ral-1/extracellular signal-related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic beta-cell.RESEARCH DESIGN AND METHODS-These studies were carried out in INS 832/13 cells and normal rat islets. Molecular biological (e.g., overexpression or small interfering RNA [siRNA]-mediated knockdown) and pharmacologic approaches were used to determine roles for farnesylation in glucosemediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Racl activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay.RESULTS-Coprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase beta-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Racl activation and insulin secretion. Pharmacologic inhibition of Raf-1 kinase using GW-5074 markedly reduced the stimulatory effects of glucose on ERK1/2 phosphorylation, Rac1 activation, and insulin secretion, suggesting that Raf-1 kinase activation may be upstream to ERK1/2 and Racl activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Racl activation and insulin secretion.CONCLUSIONS-Together, our findings provide the first evidence of a role for protein farnesylation in glucose-mediated regulation of the Raf/ERK signaling pathway culminating in the activation of Rac1, which has been shown to be necessary for cytoskeletal reorganization and exocytotic secretion of insulin. Diabetes 59:967-977, 2010