Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, Michigan, USA.
Diabetes. 2010 Apr;59(4):967-77. doi: 10.2337/db09-1334. Epub 2010 Jan 13.
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 Raf-1/extracellular signal-related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic beta-cell.
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 glucose-mediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Rac1 activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay.
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 Rac1 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 Rac1 activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Rac1 activation and insulin secretion.
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.
小 G 蛋白的翻译后棕榈酰化（例如法尼基化）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，我们研究了蛋白质法尼基化在葡萄糖诱导的 Rac1 激活和胰腺β细胞胰岛素分泌中 Raf-1/细胞外信号相关激酶 (ERK1/2) 信号通路相关信号步骤中的作用。
这些研究在 INS 832/13 细胞和正常大鼠胰岛中进行。采用分子生物学（例如过表达或小干扰 RNA [siRNA]介导的敲低）和药理学方法来确定法尼基化在葡萄糖介导的 ERK1/2、Rac1 和胰岛素分泌激活中的作用。通过 Western blot 测定 ERK1/2 的激活。通过 p21 激活激酶下拉测定法定量 Rac1 激活（即 Rac1.GTP）。通过酶联免疫吸附测定法定量胰岛素释放。
共提供法尼基转移酶（FTase；例如 FTI-277 或 FTI-2628）的结构特异性抑制剂或 siRNA 介导的 FTase β亚单位敲低，可显著抑制葡萄糖刺激的 ERK1/2 和 Rac1 激活和胰岛素分泌。使用 GW-5074 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，表明 Raf-1 激酶激活可能在上游激活 ERK1/2 和 Rac1 激活，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 Rac1 激活和胰岛素分泌。
总之，我们的研究结果首次提供了证据表明蛋白质法尼基化在葡萄糖介导的 Raf/ERK 信号通路调节中发挥作用，最终导致 Rac1 的激活，这已被证明是细胞骨架重排和胰岛素分泌的必需条件。
