Cao Hong, Schroeder Barbara, Chen Jing, Schott Micah B, McNiven Mark A
From the Department of Biochemistry and Molecular Biology, Center for Basic Research in Digestive Diseases, and.
Department of Experimental Pathology and Laboratory Medicine, Mayo Clinic, Rochester, Minnesota 55905.
J Biol Chem. 2016 Aug 5;291(32):16424-37. doi: 10.1074/jbc.M116.724997. Epub 2016 May 24.
Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments.
网格蛋白介导的转铁蛋白（Tf）及其同源受体（TfR1）的内吞作用是支持营养性铁摄取的核心途径。一般认为这是一个组成性过程。然而，我们曾报道非受体酪氨酸激酶Src可被Tf激活，以促进Tf-TfR1配体-受体复合物的内化。作为这些发现的延伸，我们测试了后续的运输步骤是否可能受其他激酶依赖性级联反应的调节，并且我们通过多种方法抑制c-Abl激酶观察到了显著的内吞阻断。重要的是，在所有这些细胞模型中，Tf内化均显著降低，并且通过重新表达野生型c-Abl可使其恢复。令人惊讶的是，这种减弱的Tf-TfR1内吞作用是由于表面和总细胞受体水平均大幅下降所致。对低密度脂蛋白受体的进一步研究显示了类似的效果。令人惊讶的是，对伊马替尼处理的细胞进行免疫荧光显微镜检查发现，内化的TfR1与晚期内体/溶酶体有明显的共定位，而用几种抑制剂减弱溶酶体功能可减少这种受体损失。重要的是，抑制c-Abl导致伴侣蛋白Hsc70从弥漫性的胞质定位显著重新分布至晚期内体-溶酶体处与TfR1结合。药物VER155008对培养细胞中Hsc70 ATP酶活性的药理学抑制作用可阻止这种伴侣蛋白-受体相互作用，导致TfR1在早期内体中积累。因此，抑制c-Abl可使受体再循环途径最小化，并导致伴侣蛋白依赖性地将TfR1运输至溶酶体进行降解。这些发现暗示了c-Abl和Hsc70作为Hsc70介导的营养性受体货物在早期和晚期内体区室之间运输的意外调节因子的新作用。
