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Development of FRET Biosensors to Detect Kinase Activity in Living Cells
Author(s) -
Okáľová Jennifer,
Burton Jeremy,
Grimsey Neil J.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04036
Subject(s) - förster resonance energy transfer , microbiology and biotechnology , g protein coupled receptor , autophosphorylation , yellow fluorescent protein , signal transduction , protein kinase a , p38 mitogen activated protein kinases , phosphorylation , biology , chemistry , biochemistry , fluorescence , physics , quantum mechanics , gene
G‐protein‐coupled receptors (GPCRs) are a group of over 800 membrane receptors essential for pathological proinflammatory signaling. The p38 mitogen‐activated protein kinase (MAPK) pathway plays a crucial role in inflammatory responses found in many diseases. Anti‐inflammatory therapeutics targeting p38 have been widely underwhelming in clinical trials, mostly due to the broad physiological and pathophysiological pathways controlled by p38. Our group discovered proinflammatory GPCRs that are associated with an atypical pathway for p38 activation in vascular cells. This atypical pathway requires transforming growth factor‐β (TGFβ) activated kinase‐1 binding protein‐1 (TAB1) to bind directly to p38, ultimately inducing p38 auto‐activation via autophosphorylation. Atypical p38 signaling provides a mechanism to selectively target pathological p38 signaling. We are developing a fluorescent biosensor platform to assess the spatiotemporal activation of atypical p38 signaling using subcellular targeted fluorescence resonance energy transfer (FRET) biosensors, which consist of two fluorescent proteins, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), joined by a linker peptide that changes conformation upon phosphorylation by p38. These FRET biosensors enable real‐time detection of p38 kinase activity at specific organelles by measuring changes in fluorescence ratios. When in close proximity, CFP will donate energy to YFP, changing the mean fluorescence from 485 to 527 nm. Through the use of endosomal, nuclear, plasma membrane, or diffuse/cytosolic targeting motifs, we have characterized specific variances in spatiotemporal GPCR dependent activation of p38. These studies represent a fundamental advancement to our knowledge of p38 activation. Additionally, future expansion of this platform into stable cell lines will enable the screening of small‐molecule inhibitors of the pathway to regulate the progression of vascular inflammation.