Fluorescence Resonance Energy Transfer (FRET) Spatiotemporal Mapping of Atypical P38 Reveals an Endosomal and Cytosolic Spatial Bias

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<b>Abstract ID 29896</b> <b>Poster Board 349</b> Mitogen-activated protein kinase (MAPK) p38 is a central regulator of intracellular signaling, driving physiological and pathological pathways. With over 150 downstream targets, it is predicted that spatial positioning and the availability of cofactors and substrates determine kinase signaling specificity. The subcellular localization of classical mitogen-activated kinase kinase 3/6 (MKK3/6) dependent p38 is highly dynamic to facilitate the selective activation of spatially restricted substrates displaying rapid nuclear translocation. In addition to classical MKK3/6-dependent p38 activation, the adaptor protein TAB1 can selectively bind to p38, inducing p38 autophosphorylation in a pathway termed atypical p38 activation. Recent studies have linked atypical p38 activity to a wide range of pathological signaling responses, including vascular inflammation and ischemic damage. However, the spatiotemporal profile of atypical p38 inflammatory signaling has not been studied and would provide valuable insight into how atypical p38-driven pathological responses. To address this critical gap, we developed genetically encoded fluorescence resonance energy transfer (FRET) biosensors to track p38 activity with subcellular resolution. Through comparative analysis of plasma membrane, cytosolic, nuclear, and endosomal compartments, we established a characteristic profile of nuclear bias for MKK3/6-dependent p38 activation. Conversely, atypical p38 activation via thrombin-mediated G-protein coupled receptor (GPCR) protease-activated receptor 1 (PAR1) activity led to the sequestration of p38 at the endosome and cytosol, limiting nuclear translocation, a profile conserved for additional inflammatory GPCRs, including prostaglandin E2 activation of EP2. Intriguingly, perturbation of GPCR endocytosis with the dynamin inhibitor Dyngo4A led to spatiotemporal switching of atypical signaling, reducing endosomal and cytosolic p38 activation and increasing nuclear activity. Suggesting that a critical and yet unknown endosomal regulator drives the spatial bias of atypical p38 signaling. The data presented provide the first live-cell examination of the spatiotemporal dynamics for p38 activity and provide critical insight into how atypical p38 signaling drives differential signaling responses through spatial sequestration of kinase activity. JB supported in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002378 and TL1TR002382, and NG, JB Supported in part by NIH NIAID R03AI171967-01