Active mutants of the TCR-mediated p38α alternative activation site show changes in the phosphorylation lip and DEF site formation

Netanel Tzarum; Ron Diskin; David Engelberg; Oded Livnah

doi:10.1016/j.jmb.2010.11.023

Active mutants of the TCR-mediated p38α alternative activation site show changes in the phosphorylation lip and DEF site formation

Netanel Tzarum, Ron Diskin, David Engelberg, Oded Livnah^*

^*Corresponding author for this work

Department of Biological Chemistry

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

The p38α mitogen-activated protein kinase is commonly activated by dual (Thr and Tyr) phosphorylation catalyzed by mitogen-activated protein kinase kinases. However, in T-cells, upon stimulation of the T-cell receptor, p38α is activated via an alternative pathway, involving its phosphorylation by zeta-chain-associated protein kinase 70 on Tyr323, distal from the phosphorylation lip. Tyr323-phosphorylated p38α is autoactivated, resulting in monophosphorylation of Thr180. The conformational changes induced by pTyr323 mediating autoactivation are not known. The lack of pTyr323 p38α for structural studies promoted the search for Tyr323 mutations that may functionally emulate its effect when phosphorylated. Via a comprehensive mutagenesis of Tyr323, we identified mutations that rendered the kinase intrinsically active and others that displayed no activity. Crystallographic studies of selected active (p38α^Y323Q, p38α ^Y323T, and p38α^Y323R) and inactive (p38α^Y323F) mutants revealed that substantial changes in interlobe orientation, extended conformation of the activation loop, and formation of substrate docking DEF site (docking site for extracellular signal-regulated kinase FXF) interaction pocket are associated with p38α activation.

Original language	English
Pages (from-to)	1154-1169
Number of pages	16
Journal	Journal of Molecular Biology
Volume	405
Issue number	5
DOIs	https://doi.org/10.1016/j.jmb.2010.11.023
State	Published - 4 Feb 2011

Bibliographical note

Funding Information:
This research was supported by the Israel Science Foundation grant 630/07 and Israel Science Foundation Research Center of Excellence 180/09 awarded to O.L. and D.E. We would like to thank Dr. Mario Lebendiker from the Protein Purification Unit of the Wolfson Centre for Applied Structural Biology for his continuous helpful advice and assistance and to Dr. Deborah E. Shalev for help in constructing Fig. 5 e and for insightful discussions. We would like to thank the staff of ESRF (Grenoble, France) for their outstanding help and for maintaining and upgrading the facility.

Keywords

MAP kinase
T-cells
alternative activation
p38α
signaling

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10.1016/j.jmb.2010.11.023

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title = "Active mutants of the TCR-mediated p38α alternative activation site show changes in the phosphorylation lip and DEF site formation",

abstract = "The p38α mitogen-activated protein kinase is commonly activated by dual (Thr and Tyr) phosphorylation catalyzed by mitogen-activated protein kinase kinases. However, in T-cells, upon stimulation of the T-cell receptor, p38α is activated via an alternative pathway, involving its phosphorylation by zeta-chain-associated protein kinase 70 on Tyr323, distal from the phosphorylation lip. Tyr323-phosphorylated p38α is autoactivated, resulting in monophosphorylation of Thr180. The conformational changes induced by pTyr323 mediating autoactivation are not known. The lack of pTyr323 p38α for structural studies promoted the search for Tyr323 mutations that may functionally emulate its effect when phosphorylated. Via a comprehensive mutagenesis of Tyr323, we identified mutations that rendered the kinase intrinsically active and others that displayed no activity. Crystallographic studies of selected active (p38αY323Q, p38α Y323T, and p38αY323R) and inactive (p38αY323F) mutants revealed that substantial changes in interlobe orientation, extended conformation of the activation loop, and formation of substrate docking DEF site (docking site for extracellular signal-regulated kinase FXF) interaction pocket are associated with p38α activation.",

keywords = "MAP kinase, T-cells, alternative activation, p38α, signaling",

author = "Netanel Tzarum and Ron Diskin and David Engelberg and Oded Livnah",

note = "Funding Information: This research was supported by the Israel Science Foundation grant 630/07 and Israel Science Foundation Research Center of Excellence 180/09 awarded to O.L. and D.E. We would like to thank Dr. Mario Lebendiker from the Protein Purification Unit of the Wolfson Centre for Applied Structural Biology for his continuous helpful advice and assistance and to Dr. Deborah E. Shalev for help in constructing Fig. 5 e and for insightful discussions. We would like to thank the staff of ESRF (Grenoble, France) for their outstanding help and for maintaining and upgrading the facility. ",

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AU - Diskin, Ron

AU - Engelberg, David

AU - Livnah, Oded

N1 - Funding Information: This research was supported by the Israel Science Foundation grant 630/07 and Israel Science Foundation Research Center of Excellence 180/09 awarded to O.L. and D.E. We would like to thank Dr. Mario Lebendiker from the Protein Purification Unit of the Wolfson Centre for Applied Structural Biology for his continuous helpful advice and assistance and to Dr. Deborah E. Shalev for help in constructing Fig. 5 e and for insightful discussions. We would like to thank the staff of ESRF (Grenoble, France) for their outstanding help and for maintaining and upgrading the facility.

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N2 - The p38α mitogen-activated protein kinase is commonly activated by dual (Thr and Tyr) phosphorylation catalyzed by mitogen-activated protein kinase kinases. However, in T-cells, upon stimulation of the T-cell receptor, p38α is activated via an alternative pathway, involving its phosphorylation by zeta-chain-associated protein kinase 70 on Tyr323, distal from the phosphorylation lip. Tyr323-phosphorylated p38α is autoactivated, resulting in monophosphorylation of Thr180. The conformational changes induced by pTyr323 mediating autoactivation are not known. The lack of pTyr323 p38α for structural studies promoted the search for Tyr323 mutations that may functionally emulate its effect when phosphorylated. Via a comprehensive mutagenesis of Tyr323, we identified mutations that rendered the kinase intrinsically active and others that displayed no activity. Crystallographic studies of selected active (p38αY323Q, p38α Y323T, and p38αY323R) and inactive (p38αY323F) mutants revealed that substantial changes in interlobe orientation, extended conformation of the activation loop, and formation of substrate docking DEF site (docking site for extracellular signal-regulated kinase FXF) interaction pocket are associated with p38α activation.

AB - The p38α mitogen-activated protein kinase is commonly activated by dual (Thr and Tyr) phosphorylation catalyzed by mitogen-activated protein kinase kinases. However, in T-cells, upon stimulation of the T-cell receptor, p38α is activated via an alternative pathway, involving its phosphorylation by zeta-chain-associated protein kinase 70 on Tyr323, distal from the phosphorylation lip. Tyr323-phosphorylated p38α is autoactivated, resulting in monophosphorylation of Thr180. The conformational changes induced by pTyr323 mediating autoactivation are not known. The lack of pTyr323 p38α for structural studies promoted the search for Tyr323 mutations that may functionally emulate its effect when phosphorylated. Via a comprehensive mutagenesis of Tyr323, we identified mutations that rendered the kinase intrinsically active and others that displayed no activity. Crystallographic studies of selected active (p38αY323Q, p38α Y323T, and p38αY323R) and inactive (p38αY323F) mutants revealed that substantial changes in interlobe orientation, extended conformation of the activation loop, and formation of substrate docking DEF site (docking site for extracellular signal-regulated kinase FXF) interaction pocket are associated with p38α activation.

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Active mutants of the TCR-mediated p38α alternative activation site show changes in the phosphorylation lip and DEF site formation

Abstract

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Keywords

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