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Identification of serine/threonine-specific phosphatases that dephosphorylate the co-chaperone BAG3 to mediate protein-protein interactions

ABSTRACT: AP-MS study of the human flag-tagged co-chaperone BAG3 to identify putative phosphatases which can regulate BAG3 function. From the two hits PP1 and PP5, the later was confirmed in a reverse AP-MS experiment. PLEASE NOTE THAT ONLY THE FILES LABELLED 'CORRECTED' BELONG TO THIS DATASET

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell, Embryonic Stem Cell, Kidney

SUBMITTER: Julian Bender

LAB HEAD: Bettina Warscheid

PROVIDER: PXD050035 | Pride | 2024-11-05

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	Flag-Bag3_iRT.fasta	Fasta
	QEplus035774.raw	Raw
	QEplus035775.raw	Raw
	QEplus035776.raw	Raw
	QEplus035778.raw	Raw

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Publications

Identification of phosphatases that dephosphorylate the co-chaperone BAG3.

Kokot Thomas T Zimmermann Johannes P JP Chand Yamini Y Krier Fabrice F Reimann Lena L Scheinost Laura L Höfflin Nico N Esch Alessandra A Höhfeld Jörg J Warscheid Bettina B Köhn Maja M

Life science alliance 20241119 2

The co-chaperone BAG3 plays critical roles in maintaining cellular proteostasis. It associates with 14-3-3 proteins during the trafficking of aggregation-prone proteins and facilitates their degradation through chaperone-assisted selective autophagy in cooperation with small heat shock proteins. Although reversible phosphorylation regulates BAG3 function, the involved phosphatases remain unknown. Here, we used affinity purification mass spectrometry to identify phosphatases that target BAG3. Of ...[more]

PMID: 39562141

Similar Datasets

Identification of serine/threonine-specific phosphatases that dephosphorylate the co-chaperone BAG3 to mediate protein-protein interactions

Project description:Targeted phosphoproteomics of the human flag-tagged co-chaperone BAG3 to quantify phosphosite regulation by PP5.

2024-11-05 | PXD050006 | Pride

Identification of serine/threonine-specific phosphatases that dephosphorylate the co-chaperone BAG3 to mediate protein-protein interactions

Project description:AP-MS study of the human flag-tagged co-chaperone BAG3 and phospho-mimicking mutants (D/A) to identify phosphorylation-dependent changes in binding.

2024-11-05 | PXD057392 | Pride

Overexpression of the human mutation P209L of cochaperone BAG3 in mouse

Project description:An amino acid exchange (P209L) in the HSPB8 binding site of the human cochaperone Bcl2-associated athanogene 3 (BAG3) gives rise to severe dominant childhood cardiomyopathy. To phenocopy the disease in mouse and gain insight into its mechanisms, we have generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates containing BAG3, components of the Z-disc, and the protein quality control system in cardiomyocytes. This was accompanied by massive fibrosis resulting in a severe, early-onset restrictive cardiomyopathy with increased mortality, as observed in patients. Here we present the shotgun proteome data of mice expressing hBAG3P209L-eGFP compared to control.

2021-04-13 | PXD021165 | Pride

SUMOylation of Protein Phosphatase 5 Regulates Phosphatase Activity and Substrate Release

Project description:The serine/threonine protein phosphatase 5 (PP5) regulates hormone and stress-induced signaling networks. PP5 has been suggested to exist largely in a ‘closed’ and autoinhibitory state whereas the ‘open’ conformation is required for activity. Additionally, PP5 is subject to post-translational modification (PTMs), however, the impact of PTMs on PP5 conformation, activity, and substrate release in cells remains elusive. Here we identified that SUMOylation of K430 in the catalytic domain of PP5 facilitates substrate release. Phosphorylation of PP5-T362 and activation is pre-requisite for SUMOylation suggesting the ordered addition of PTMs carefully regulates PP5 function in cells. Additionally, our quantitative Förster Resonance Energy Transfer (FRET) in live mammalian cells showed that phosphorylation and SUMOylation of PP5 favor a ‘closed’ conformation that functionally is quite different from the autoinhibitory state of PP5. This suggests that PP5 samples multiple conformational states within a cell as a result of PTMs, which do not necessarily correlate with PP5 activity. Furthermore, comparative proteomic analysis of PP5-WT and K430R identified many new potential substrates of PP5. Taken together, our data presented here demonstrate a crucial regulatory step of PP5 activity, SUMOylation of K430R. Notably, the characteristics of a non-SUMOylatable PP5 mutant, K430R, allow rapid identification of new PP5 substrates.

2023-09-08 | PXD038701 | Pride

Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Project description:Objective DLL1 is a mammalian Notch ligand with essential functions during embryonic development. We tagged endogenous DLL1 in one homologous recombination step such that AcGFP-HA tagged DLL1 could be converted by Cre-mediated site-specific recombination into StrepFlag tagged DLL1. We anticipated that this should allow us to visualise DLL1 in living cells as well as allow for sorting and enrichment of DLL1-expressing cells and efficient purification of DLL1 protein complex. Results We generated constructs to express a DLL1 variant that carried C-terminal in frame an AcGFPHA tag flanked by loxP sites followed by a StrepFlag tag out of frame. Cre-mediated recombination removed AcGFP-HA and added StrepFlag in frame to DLL1. The AcGFPHAstopStrepFlag tag was added to the Dll1 cDNA to allow for tests in cultured cells in vitro and was introduced into endogenous DLL1 in mice using ES cells modified by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cell and embryo lysates. In CHO cells the AcGFP protein fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the StrepFlag tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or StrepFlag-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

2021-11-03 | PXD024680 | Pride

BAG3 regulates cilia homeostasis of glioblastoma via its WW domain

Project description:The multidomain protein BAG3 exerts pleiotropic oncogenic functions in many tumor entities including glioblastoma (GBM). Here we compared BAG3 protein-protein interactions in either adherently cultured or stem-like cultured U251 GBM cells. In line with BAG3´s putative role in stemness, identified interactors in sphere-cultured cells included different stem cell markers (SOX2, OLIG2, NES), while interactomes of adherent BAG3-proficient cells indicated a shift towards involvement of BAG3 in regulation of cilium assembly (ACTR3, ARL3). Applying a set of BAG3 deletion constructs we could demonstrate that none of the domains except the WW domain are required for suppression of cilia formation by full-length BAG3 in U251 and U343 cells. In line with the established regulation of the Hippo pathway by this domain, we could show that the WW mutant fails to rescue YAP1 nuclear translocation. BAG3 depletion reduced activation of a YAP1/AURKA signaling pathway and induction of PLK1. Collectively, our findings point to a complex interaction network of BAG3 with several pathways regulating cilia homeostasis, involving processes related to ciliogenesis and cilium degradation.

2024-04-09 | PXD050279 | Pride

Flnc d18-21/d1-3 pulldown experiments

Project description:The actin-binding protein filamin c (FLNc) is a key mediator in the response of skeletal muscle cells to mechanical stress. In addition to its function as a structural scaffold, FLNc acts as a signaling adaptor which is phosphorylated at S2234 in its mechanosensitive domain 20 (d20) through AKT. Here, we discovered a strong dephosphorylation of FLNc-pS2234 in skeletal myotubes under acute mechanical stress, despite high AKT activity. We found that all three protein phosphatase 1 (PP1) isoforms are part of the FLNc d18-21 interactome. Enzymatic assays demonstrate that PP1 efficiently dephosphorylates FLNc-pS2234 in vitro and in cells upon PP1 activation using specific modulators. FLNc-pS2234 dephosphorylation promotes the interaction with FILIP1, a mediator for filamin degradation. Collectively, we present a model in which dephosphorylation of FLNc d20 by the dominant action of PP1c prevails over AKT activity to promote the binding of the filamin degradation-inducing factor FILIP1 during acute mechanical stress. Note that mouse FLNc S2234/S2237 correspond to S2233 and S2236 in human FLNc.

2025-05-07 | PXD053236 | Pride

Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

Project description:Loss of Bcl2-associated anthanogene 3 (BAG3) is associated with dilated cardiomyopathy (DCM). Recent studies show that BAG3 regulates sarcomere protein turnover in cardiomyocytes (CMs). However, the function of BAG3 in other cell types of the heart is unknown. In this study, we used an isogenic pair of BAG3 knockout and wild-type human induced pluripotent stem cells (hiPSC) to interrogate the function of BAG3 in hiPSC-derived cardiac fibroblasts (CFs). Generating a series of conditional knockout (cKO) engineered heart tissues, we determined the contribution of CF BAG3 to engineered heart tissue function. Despite normal CM genotype, the loss of fibroblast-specific BAG3 (FBKO) caused a reduction in contractile force and an increase in cardiac fibrosis in the engineered heart tissues, recapitulating the phenotype of DCM. Using culture substrates with defined stiffness, we decoupled the mechanical activation of fibroblasts from their ligand response. In BAG3-/- CFs, we observed an increased sensitivity to transforming growth factor β (TGFβ) signaling and activation of a fibrogenic response when cultured at physiological stiffness (8kPa). Mechanistically, loss of BAG3 increased transforming growth factor receptor 2 (TGFBR2) levels. BAG3 binds TGFBR2 and mediates its ubiquitination and proteasomal degradation. To further validate these results, we performed single-nuclei sequencing of cardiac tissue from DCM patients carrying pathogenic BAG3 mutations. Mutations in BAG3 increased fibrotic gene expression in cardiac fibroblasts. Together, these results extend our understanding of the roles of BAG3 in heart disease beyond the cardiomyocyte-centric view. This study also highlights the ability of hiPSC-based models and tissue engineering to answer questions about the cell-type specific aspects of cardiac disease.

2024-09-16 | GSE261750 | GEO

Calpain Cleavage of Junctophilin-2 generates a Spectrum of Calcium-dependent Cleavage Products and DNA-rich NT1-Fragment Domains in Cardiomyocytes - PRM Dataset

Project description:Calpains are calcium (Ca2+) activated neutral proteases that are involved in several essential signaling pathways. One Calpain-specific proteolytic target is Junctophilin-2 (JP2), an important structural protein of Ca2+ release units required for the excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in mouse models of heart failure has been reported previously, the precise molecular identity of the Calpain cleavage sites and the (patho-) physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565, but subsequently also at three additional secondary Calpain cleavage sites. We identified the Calpain-specific primary cleavage products not only in mouse but also in human iPSC-derived cardiomyocytes (hiPSC-CMs). Knockout of RyR2 in hiPSC-CMs destabilized JP2 resulting in an increase of the Calpain-specifc cleavage fragments. The primary N-terminal cleavage product (NT1) accumulated in the nucleus of mouse and human cardiomyocytes in a Ca2+ dependent manner, closely associated with DNA-rich chromosomal regions, where NT1 is proposed to function as a cardioprotective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT1 by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.

2022-08-12 | PXD031424 | Pride

Proteome analysis of pathogen-induced regulated cell death in maize

Project description:Regulated cell death (RCD) is crucial for plant development, as well as in decision-making in plant-pathogen interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. We analyzed the proteome of Z. mays leaves treated with the effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time-dependent biological processes being activated on proteome level in response to avrRxo1, FB1 and SA. This study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death.

2023-07-13 | PXD041377 | Pride

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