Epidermal growth factor-stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis.
Epidermal growth factor-stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis.
复制标题
表皮生长因子刺激的Akt磷酸化需要网格蛋白或ERBB2，但不需要受体内吞作用。
DOI：
10.1091/mbc.e14-09-1412
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发表时间：
2015-10-01
影响因子：
3.3
通讯作者：
Antonescu CN
中科院分区：
文献类型：
--
作者：
Garay C;Judge G;Lucarelli S;Bautista S;Pandey R;Singh T;Antonescu CN
配体结合后，表皮生长因子受体 (EGFR) 激活信号传导并进行内吞作用。导致 Akt 激活的 EGFR 信号传导会因网格蛋白的扰动而受损，但不会因动力扰动而抑制内化而受损。因此，网格蛋白可以直接调节细胞表面的受体信号传导。表皮生长因子 (EGF) 与其受体 (EGFR) 结合可激活多种信号传导中间体，包括 Akt，从而控制细胞存活和代谢。与此同时，配体结合的 EGFR 被纳入网格蛋白包被的凹坑（含有网格蛋白和其他蛋白质的膜结构）中，最终导致受体内化。网格蛋白是否可能在囊泡分裂前调节质膜上的 EGFR 信号转导尚不清楚。我们在 EGFR 内吞作用依赖于网格蛋白的条件下，比较了网格蛋白扰动（防止网格蛋白结构的形成或受体募集到网格蛋白结构）与 dynamin2（允许网格蛋白结构的形成但防止 EGFR 内化）的效果。通过 siRNA 基因沉默、网格蛋白抑制剂 pitstop2 或 knocksideways 沉默来干扰网格蛋白，可抑制 ARPE-19 细胞中 EGF 模拟的 Gab1 和 Akt 磷酸化。相反，用抑制剂或 siRNA 基因沉默扰动 dynamin2 不会影响 EGF 刺激的 Gab1 或 Akt 磷酸化。 EGF 刺激使网格蛋白结构内的 Gab1 和磷酸化 Gab1 富集。 ARPE-19 细胞具有较低的 ErbB2 表达，过表达和敲低实验表明，强大的 ErbB2 表达绕过了 EGF 刺激的 Akt 磷酸化对网格蛋白的需求。因此，网格蛋白支架可能代表某些受体信号传导所需的独特质膜信号传导微结构域，该功能可以与囊泡形成分开。
Upon ligand binding, the epidermal growth factor receptor (EGFR) activates signaling and undergoes endocytosis. EGFR signaling leading to Akt activation is impaired by perturbation of clathrin but not by inhibition of internalization through perturbation of dynamin. Clathrin may thus directly regulate receptor signaling at the cell surface. Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.