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

Mitogen-activated protein (MAP) kinase signaling is central to multiple cellular responses and processes. MAP kinase p38 alpha is the best characterized member of the p38 MAP kinase family. Upstream factors and downstream targets of p38 alpha 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 p38 alpha in cultured 293T cells. Fusing the advanced biotin ligase BioID2 to the N-terminus of p38 alpha, we used mass spectrometry to identify 37 biotin-labeled proteins that putatively interact with p38 alpha. 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 p38 alpha in cells supports an interaction of p38 alpha 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 p38 alpha in cells and new direct molecular targets of MAP kinase p38 alpha in gene regulation and the DNA damage response.