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Proc Natl Acad Sci U S A. 2019 Jan 15;116(3):810-815. doi: 10.1073/pnas.1819230116. Epub 2018 Dec 27.

Arrestin-3 scaffolding of the JNK3 cascade suggests a mechanism for signal amplification.

Author information

1
Department of Pharmacology, Vanderbilt University, Nashville, TN 32232.
2
Division of Chemical Biology & Medicinal Chemistry, The University of Texas at Austin, Austin, TX 78712.
3
Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt.
4
Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN 32232.
5
Department of Psychiatry and Behavioral Sciences, Vanderbilt University, Nashville, TN 32232.
6
Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN 32232.
7
Department of Chemistry, Tennessee Technological University, Cookeville, TN 38505.
8
Department of Pharmacology, Vanderbilt University, Nashville, TN 32232; c.lopez@vanderbilt.edu tina.iverson@vanderbilt.edu vsevolod.gurevich@vanderbilt.edu.
9
Department of Biochemistry, Vanderbilt University, Nashville, TN 32232.
10
Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 32232.
11
Department of Bioinformatics, Vanderbilt University, Nashville, TN 32232.
12
Center for Structural Biology, Vanderbilt University, Nashville, TN 32232.

Abstract

Scaffold proteins tether and orient components of a signaling cascade to facilitate signaling. Although much is known about how scaffolds colocalize signaling proteins, it is unclear whether scaffolds promote signal amplification. Here, we used arrestin-3, a scaffold of the ASK1-MKK4/7-JNK3 cascade, as a model to understand signal amplification by a scaffold protein. We found that arrestin-3 exhibited >15-fold higher affinity for inactive JNK3 than for active JNK3, and this change involved a shift in the binding site following JNK3 activation. We used systems biochemistry modeling and Bayesian inference to evaluate how the activation of upstream kinases contributed to JNK3 phosphorylation. Our combined experimental and computational approach suggested that the catalytic phosphorylation rate of JNK3 at Thr-221 by MKK7 is two orders of magnitude faster than the corresponding phosphorylation of Tyr-223 by MKK4 with or without arrestin-3. Finally, we showed that the release of activated JNK3 was critical for signal amplification. Collectively, our data suggest a "conveyor belt" mechanism for signal amplification by scaffold proteins. This mechanism informs on a long-standing mystery for how few upstream kinase molecules activate numerous downstream kinases to amplify signaling.

KEYWORDS:

arrestin; cell signaling; mitogen-activated protein kinase; protein scaffold; signal amplification

PMID:
30591558
PMCID:
PMC6338856
DOI:
10.1073/pnas.1819230116
[Indexed for MEDLINE]
Free PMC Article

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