Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K),...

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Published in	Proceedings of the National Academy of Sciences - PNAS Vol. 118; no. 37; pp. 1 - 11
Main Authors	Qu, Changxiu, Park, Ji Young, Yun, Min Woo, He, Qing-tao, Yang, Fan, Kim, Kiae, Ham, Donghee, Li, Rui-rui, Iverson, T. M., Gurevich, Vsevolod V., Sun, Jin-Peng, Chung, Ka Young
Format	Journal Article
Language	English
Published	United States National Academy of Sciences 14.09.2021
Subjects	Animals Arrestin Arrestins - metabolism beta-Arrestin 1 - metabolism beta-Arrestin 2 - metabolism beta-Arrestins - metabolism Binding Biological Sciences Chlorocebus aethiops COS Cells Desensitization Deuterium Extracellular signal-regulated kinase Extracellular Signal-Regulated MAP Kinases - metabolism Fluorescence Fluorescence Resonance Energy Transfer - methods Homology Humans Hydrogen-deuterium exchange Interfaces Internalization Kinases MAP kinase MAP Kinase Kinase 1 - metabolism MAP Kinase Kinase Kinases - metabolism MAP Kinase Signaling System - physiology Mass spectrometry Mass Spectrometry - methods Mass spectroscopy Mitogen-Activated Protein Kinase Kinases - metabolism Mitogen-Activated Protein Kinases - metabolism NMR Nuclear magnetic resonance Nuclear Magnetic Resonance, Biomolecular - methods Phosphorylation Protein Binding Protein Processing, Post-Translational Protein Serine-Threonine Kinases Proteins Proteins - metabolism Raf protein Rats Receptors Scaffolding Signal Transduction Signaling Tryptophan MEK cRaf arrestin scaffold ERK
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ISSN	0027-8424 1091-6490 1091-6490
DOI	10.1073/pnas.2026491118

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Summary:	Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange–mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 1C.Q., J.Y.P., and M.W.Y. contributed equally to this work. Edited by David Baker, University of Washington, Seattle, WA, and approved June 28, 2021 (received for review December 24, 2020) The authors declare no competing interest. Author contributions: J.-P.S. and K.Y.C. designed research; C.Q., J.Y.P., M.W.Y., Q.-t.H., F.Y., K.K., R.-r.L., J.-P.S., and K.Y.C. performed research; J.-P.S. and K.Y.C. contributed new reagents/analytic tools; C.Q., J.Y.P., M.W.Y., K.K., D.H., J.-P.S., and K.Y.C. analyzed data; and C.Q., J.Y.P., M.W.Y., T.M.I., V.V.G., J.-P.S., and K.Y.C. wrote the paper.
ISSN:	0027-8424 1091-6490 1091-6490
DOI:	10.1073/pnas.2026491118