Regulation of the cochaperone BAG3 and chaperone-assisted selective autophagy by Hippo kinases

辅伴侣BAG3(BCL2相关的athanogene-3)是肌肉、免疫和神经细胞中的一个关键的蛋白稳定因子。此外，它的过表达有助于癌基因的转化。BAG3诱导一条伴侣辅助和泛素依赖的自噬途径，称为CASA(伴侣辅助选择性自噬)。该途径介导非本地伴侣客户的溶酶体降解，如肌动蛋白锚定蛋白细丝在收缩肌肉和贴壁细胞中机械展开后的细丝蛋白。BAG3除了具有降解诱导活性外，还调节蛋白质的合成。辅伴侣与河马信号通路的组成部分相互作用，以促进细丝蛋白的表达。BAG3介导的蛋白质降解和蛋白质合成的共同调节对于维持机械应变下的肌动蛋白细胞骨架是必不可少的。在这里，我们打算分析河马信号对BAG3功能和CASA活性的影响。该项目是基于我们对BAG3相关的多个河马蛋白激酶的鉴定。我们的初步数据显示，这些激酶中至少有一个直接控制着平滑肌细胞中的CASA活性。我们将应用生化和细胞生物学方法来验证河马蛋白激酶与肌肉和神经细胞中CASA机制之间的功能相互作用。将利用磷酸蛋白质组学来确定CASA组分中的河马磷酸化位点，并探讨它们对BAG3和CASA调控的重要性。最后，分化的骨骼肌细胞、分离的小鼠肌肉和转基因黑腹果蝇系将被用来研究Hippo信号在BAG3介导的肌肉维持中的作用。这项工作应该为真核生物中蛋白质稳态的调节建立关键机制，这与我们对肌肉弱点、神经退化和癌症的理解相关。

The cochaperone BAG3 (BCL2-associated athanogene-3) is a key proteostasis factor in muscle, immune and neuronal cells. In addition, its overexpression contributes to oncogenic transformation. BAG3 induces a chaperone-assisted and ubiquitin-dependent autophagy pathway, called CASA (chaperone-assisted selective autophagy). The pathway mediates the lysosomal degradation of nonnative chaperone clients, such as the actin-anchoring protein filamin following its mechanical unfolding in contracting muscles and adherent cells. Besides its degradation-inducing activity, BAG3 also regulates protein synthesis. The cochaperone interacts with components of the Hippo signaling pathway to facilitate filamin expression. The BAG3-mediated coregulation of protein degradation and protein synthesis is essential for maintaining the actin cytoskeleton under mechanical strain. Here we propose to analyze the impact of Hippo signaling on BAG3 function and CASA activity. The project is based on our identification of multiple Hippo kinases in association with BAG3. Our preliminary data show that at least one of these kinases directly controls CASA activity in smooth muscle cells. We will apply biochemical and cell biological assays to verify functional interactions between Hippo kinases and the CASA machinery in muscle and neuronal cells. Phosphoproteomics will be performed to identify Hippo phosphorylation sites in CASA components, and their importance for BAG3 and CASA regulation will be explored. Finally, differentiated skeletal muscle cells, isolated mouse muscles and transgenic D. melanogaster lines will be used to investigate the role of Hippo signaling in BAG3-mediated muscle maintenance. The work should establish key mechanisms for the regulation of protein homeostasis in eukaryotes, relevant for our understanding of muscle weaknesses, neurodegeneration and cancer.