Mouse anti-Human MAPK2 monoclonal antibody, clone Q35-705

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.

Upon encountering physiological cues associated with damaged or inflamed endothelium, blood platelets set forth intracellular responses to ultimately support hemostatic plug formation and vascular repair. To gain insights into the molecular events underlying platelet function, we used a combination of interactome, pathway analysis, and other systems biology tools to analyze associations among proteins functionally modified by reversible phosphorylation upon platelet activation. While an interaction analysis mapped out a relative organization of intracellular mediators in platelet signaling, pathway analysis revealed directional signaling relations around protein kinase C (PKC) isoforms and mitogen-activated protein kinases (MAPKs) associated with platelet cytoskeletal dynamics, inflammatory responses, and hemostatic function. Pathway and causality analysis further suggested that platelets activate a specific p38-MK2 axis to phosphorylate RTN4 (reticulon-4, also known as Nogo), a Bcl-xl sequestration protein and critical regulator of endoplasmic reticulum (ER) physiology. In vitro, we find that platelets drive a p38-MK2-RTN4-Bcl-xl pathway associated with the regulation of the ER and platelet phosphatidylserine exposure. Together, our results support the use of pathway tools in the analysis of omics data sets as a means to help generate novel, mechanistic, and testable hypotheses for platelet studies while uncovering RTN4 as a putative regulator of platelet cell physiological responses.