Department of Pathology, Oslo University Hospital, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
Department of Pathology, Oslo University Hospital, Oslo, Norway.
Exp Cell Res. 2018 Oct 1;371(1):139-150. doi: 10.1016/j.yexcr.2018.08.004. Epub 2018 Aug 8.
Overexpression of ErbB2 is frequent in cancer and understanding the mechanisms which regulate its expression is important. ErbB2 is considered endocytosis resistant. It has no identified ligand, but upon heterodimerization it is a potent mediator of proliferative signaling. A recent study established a role for protein kinase C (PKC) in internalization and recycling of ErbB2. We have now further investigated the molecular mechanisms involved in PKC-mediated downregulation of ErbB2. We confirm that PMA-induced PKC activation causes ErbB2 internalization, but while the Hsp90 inhibitor 17-AAG induced ErbB2 degradation, PMA had no such effect. When combined with 17-AAG, PMA had additive effect on ErbB2 internalization indicating that Hsp90 inhibition and PKC activation induce internalization by alternative mechanisms. We confirm that while 17-AAG-induced internalization was clathrin-mediated, PMA-induced internalization was clathrin independent. This difference may be explained by while both 17-AAG and PMA reduced the constitutive tyrosine phosphorylation of ErbB2, only 17-AAG induced Hsp90 dissociation, Hsp70 recruitment and ubiquitination of ErbB2. Importantly, since PMA induced internalization of ErbB2, but not dissociation of Hsp90, Hsp90 does not per se retain ErbB2 at the plasma membrane. The morphology of the compartment into which receptors are sorted upon PKC activation has not previously been identified. By immuno-electron microscopy, we show that PMA sorts ErbB2 into a complex tubulovesicular or cisternal organelle resembling a previously described endocytic recycling compartment.
ErbB2 的过表达在癌症中很常见，了解调节其表达的机制非常重要。ErbB2 被认为是内吞作用抵抗的。它没有确定的配体，但在异二聚化后，它是增殖信号的有力介质。最近的一项研究确立了蛋白激酶 C（PKC）在 ErbB2 内化和回收中的作用。我们现在进一步研究了 PKC 介导的 ErbB2 下调所涉及的分子机制。我们证实 PMA 诱导的 PKC 激活导致 ErbB2 内化，但 Hsp90 抑制剂 17-AAG 诱导 ErbB2 降解，而 PMA 则没有这种作用。当与 17-AAG 联合使用时，PMA 对 ErbB2 内化有相加作用，表明 Hsp90 抑制和 PKC 激活通过不同的机制诱导内化。我们证实，虽然 17-AAG 诱导的内化是网格蛋白介导的，但 PMA 诱导的内化是网格蛋白非依赖性的。这种差异可以解释为，虽然 17-AAG 和 PMA 都降低了 ErbB2 的组成性酪氨酸磷酸化，但只有 17-AAG 诱导了 Hsp90 的解离、Hsp70 的募集和 ErbB2 的泛素化。重要的是，由于 PMA 诱导了 ErbB2 的内化，而不是 Hsp90 的解离，Hsp90 本身并不能将 ErbB2 保留在质膜上。以前尚未确定 PKC 激活时受体分选到的隔室的形态。通过免疫电子显微镜，我们显示 PMA 将 ErbB2 分选到类似于先前描述的内吞体再循环隔室的复杂管状小泡或小窝细胞器中。
