The Hsp70–Bag3 complex modulates the phosphorylation and nuclear translocation of Hippo pathway protein Yap

Abstract

Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70–Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathway proteins, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP (also known as YAP1). In naïve cells, Bag3 recruited a complex of LATS1, YAP and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of the proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1, and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus also facilitated nuclear translocation of YAP. Interestingly, Bag3 also controlled YAP nuclear localization in response to cell density, indicating broader roles beyond proteotoxic signaling responses for Bag3 in the regulation of YAP. These data implicate Bag3 as a regulator of Hippo pathway signaling, and suggest mechanisms by which proteotoxic stress signals are propagated.

Hsp70–Bag3 Module Regulates Macrophage Motility and Tumor Infiltration via Transcription Factor LITAF and CSF1

“…Previously, LITAF was identified as one of the Bag3-interacting proteins in multiple high-throughput proteomics experiments ( , accessed on 12 August 2022). Furthermore, a yet unpublished Ph.D. thesis demonstrated that such interaction occurs via the WW domain of Bag3 [ 59 ]. Therefore, we decided to reproduce these data in a direct co-IP experiment.…”

“…Recently, we uncovered that Bag3 directly interacts with another transcription factor, a major regulator of cell polarity and cancer development—Yap [ 59 ]. Via this interaction, Bag3 controls phosphorylation and nuclear localization of Yap.…”

“…Microscopy was performed using control and Bag3 or LITAF siRNA silenced PMA differentiated THP1 cells plated in a concentration of 8 × 10 3 cells/well in a total volume of 200 µL. The assay was carried out as previously described by Baldan et al [ 59 ]. The cells were fixed with a solution of 5% formaldehyde in PBS at room temperature for 15 min, washed once with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature.…”

Cytoplasmic proteotoxicity regulates HRI-dependent phosphorylation of eIF2α via the Hsp70-Bag3 module

“…Hsp70 alone keeps HRI in a suppressed state, while binding to Hsp70 of abnormal proteins relieves this suppression and leads to activation of HRI. Maybe, it enhances interaction of HRI with its substrate eIF2a, similar to the mechanism of activation of Hippo pathway, where proteotoxic stress via Hsp70-Bag3 regulates interaction of a kinase Lats1 with its substrate Yap ( Baldan et al., 2021 ). Addition of JG-98 dissociates Hsp70 from Bag3-HRI complex and thus relieves HRI suppression by Hsp70, resulting in a stronger phosphorylation of eIF2a ( Figures 2 A and 2C).…”

“…However, mechanisms of activation of HRI by the cytosolic proteotoxicity remain unclear. We hypothesized that Hsp70-Bag3 complex that regulates responses of stress kinases to proteotoxicity ( Baldan et al., 2021 ; Meriin et al., 2018 ) may be involved in regulation of HRI.…”