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

2021 ◽  
Vol 118 (37) ◽  
pp. e2026491118
Author(s):  
Changxiu Qu ◽  
Ji Young Park ◽  
Min Woo Yun ◽  
Qing-tao He ◽  
Fan Yang ◽  
...  
Keyword(s):  
Mapk Signaling ◽  
Signaling Proteins ◽  
Structural Mechanism ◽  
Mapk Kinase ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
G Protein Coupled ◽  
Binding Interfaces ◽  
Homologous Desensitization

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.

scholarly journals Cryo–electron microscopy structure and analysis of the P-Rex1–Gβγ signaling scaffold

2019 ◽  
Vol 5 (10) ◽  
pp. eaax8855 ◽  
Author(s):  
Jennifer N. Cash ◽  
Sarah Urata ◽  
Sheng Li ◽  
Sandeep K. Ravala ◽  
Larisa V. Avramova ◽  
...  
Keyword(s):  
G Protein ◽  
Neutrophil Migration ◽  
Signaling Proteins ◽  
Pdz Domains ◽  
Exchange Mass Spectrometry ◽  
Cryo Electron Microscopy ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Carboxyl Terminal ◽  
G Protein Coupled

PIP3-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein–coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1–Gβγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gβγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gβγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gβγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.

scholarly journals Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition

2019 ◽  
Author(s):  
Isaac Fisher ◽  
Meredith Jenkins ◽  
Greg Tall ◽  
John E Burke ◽  
Alan V. Smrcka
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
G Protein Coupled Receptors ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
G Protein Coupled ◽  
Inhibitory Interactions

AbstractPhospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαq and/or Gβγ subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLCβ3 bound to Gαq is known, however, for both Gαq and Gβγ, the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLCβ hydrolase activity.

Conformational Dynamics Analysis of MEK1 Using Hydrogen/Deuterium Exchange Mass Spectrometry

2020 ◽  
Vol 27 ◽  
Author(s):  
Min Woo Yun ◽  
Kiae Kim ◽  
Ji Young Park ◽  
Ka Young Chung
Keyword(s):  
Mass Spectrometry ◽  
Conformational Dynamics ◽  
Deuterium Exchange ◽  
Full Length ◽  
Atp Binding ◽  
Hydrogen Deuterium Exchange ◽  
Mapk Kinase ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

Background: Activation of mitogen-activated protein kinases (MAPKs) is regulated by a phosphorylation cascade comprising three kinases, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. MAP2K1 and MAPK2K2, also known as MEK1 and MEK2, activate ERK1 and ERK2. The structure of the MAPK signaling cascade has been studied, but high-resolution structural studies of MAP2Ks have often focused on kinase domains or docking sites, but not on full-length proteins. Objective: To understand the conformational dynamics of MEK1. Methods: Full-length MEK1 was purified from Escherichia coli (BL21), and its conformational dynamics were analyzed using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The effects of ATP binding were examined by coincubating MEK1 and adenylyl-imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analog. Results: MEK1 exhibited mixed EX1/EX2 HDX kinetics within the N-terminal tail through β1, αI, and the C-terminal helix. AMP-PNP binding was found to reduce conformational dynamics within the glycine-rich loop and regions near the DFG motif, along with the activation lip. Conclusion: We report for the first time that MEK1 has regions that slowly change its folded and unfolded states (mixed EX1/EX2 kinetics) and also report the conformational effects of ATP-binding to MEK1.

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