Mapping p38α mitogen‐activated protein kinase signaling by proximity‐dependent labeling

Abstract

Mitogen-activated protein (MAP) kinase signaling is central to multiple cellular responses and processes. MAP kinase p38α is the best characterized member of the p38 MAP kinase family. Upstream factors and downstream targets of p38α have been identified in the past by conventional methods such as coimmunoprecipitation. However, a complete picture of its interaction partners and substrates in cells is lacking. Here, we employ a proximity-dependent labeling approach using biotinylation tagging to map the interactome of p38α in cultured 293T cells. Fusing the advanced biotin ligase BioID2 to the N-terminus of p38α, we used mass spectrometry to identify 37 biotin-labeled proteins that putatively interact with p38α. Gene ontology analysis confirms known upstream and downstream factors in the p38 MAP kinase cascade (e.g., MKK3, MAPKAPK2, TAB2, and c-jun). We furthermore identify a cluster of zinc finger (ZnF) domain-containing proteins that is significantly enriched among proximity-labeled interactors and is involved in gene transcription and DNA damage response. Fluorescence imaging and coimmunoprecipitation with overexpressed p38α in cells supports an interaction of p38α with ZnF protein XPA, a key factor in the DNA damage response, that is promoted by UV irradiation. These results define an extensive network of interactions of p38α in cells and new direct molecular targets of MAP kinase p38α in gene regulation and the DNA damage response. K E Y W O R D SBioID, biotinylation, interactome, MAP kinase, p38, p38α, protein-protein interaction, proximity-dependent labeling

Functions of p38 MAP Kinases in the Central Nervous System

“…This approach has been used to study function of p38α in mice using a transgenic or knock-in approach (Ren et al, 2005;Cortez et al, 2017). New biochemical methods to label protein interactions and substrate engagement facilitate mapping of entire MAP kinase signaling cascades (Prikas et al, 2020). In summary, the molecular and genetic toolbox outlined above, including inhibitor molecules, gene knockout, CRISPR, RNAi, constitutively active (CA) p38 variants, and proximity labeling, facilitated the discovery of functions of p38 kinases and will be essential for future advances toward a better understanding of p38 kinases in CNS cell types and diseases.…”

P38α MAPK Signaling—A Robust Therapeutic Target for Rab5-Mediated Neurodegenerative Disease

“…However, p38α signaling has many targets aside from this classical pathway [32] and biologic effects other than regulation of proinflammatory cytokine production. With regard to neurodegenerative diseases, a large variety of biological roles have been attributed to p38α in brain pathology which depend on the type and stage of central nervous system (CNS) disease, brain region, cell type [4,18,19,26].…”

NRF2 Regulation Processes as a Source of Potential Drug Targets against Neurodegenerative Diseases

“…The P38 MAPK family comprises several isoforms (P38α, β, γ and δ) with specific distribution and substrates. Once activated by phosphorylation, they in turn phosphorylate different proteins and transcription factors to regulate processes including inflammation, cell division, and differentiation or apoptosis [235]. P38 kinases are mainly activated by oxidative stress [236], which is a pathological hallmark of NDDs.…”