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

辅伴侣蛋白BAG 3（BCL 2-associated athanogene-3）是肌肉、免疫和神经细胞中的关键蛋白质稳定因子。此外，它的过度表达有助于致癌转化。BAG 3诱导分子伴侣辅助的和泛素依赖的自噬途径，称为CASA（分子伴侣辅助的选择性自噬）。该途径介导非天然伴侣客户的溶酶体降解，例如肌动蛋白锚定蛋白细丝蛋白在收缩肌肉和粘附细胞中机械展开后。除了其降解诱导活性，BAG 3还调节蛋白质合成。辅伴侣蛋白与Hippo信号通路的组分相互作用以促进细丝蛋白表达。BAG 3介导的蛋白质降解和蛋白质合成的协同调节对于在机械应变下维持肌动蛋白细胞骨架是必需的。在这里，我们建议分析Hippo信号传导对BAG 3功能和CASA活性的影响。该项目基于我们对与BAG 3相关的多种Hippo激酶的鉴定。我们的初步数据表明，这些激酶中至少有一种直接控制平滑肌细胞中的CASA活性。我们将应用生物化学和细胞生物学检测来验证海马激酶和CASA机制在肌肉和神经元细胞中的功能相互作用。将进行磷酸化蛋白质组学以鉴定CASA组分中的Hippo磷酸化位点，并探索它们对BAG 3和CASA调控的重要性。最后，将分化的骨骼肌细胞、分离的小鼠肌肉和转基因D.黑腹果蝇品系将用于研究Hippo信号传导在BAG 3介导的肌肉维持中的作用。这项工作应该建立真核生物中蛋白质稳态调节的关键机制，与我们对肌肉无力，神经变性和癌症的理解有关。

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.