Abstract
Tyrosine kinases couple the T cell receptor (TCR) to discrete signaling cascades, each of which is capable of inducing a distinct functional outcome. Precisely how TCR signals are channeled toward specific targets remains unclear. TCR stimulation triggers 'alternative' activation of the mitogen-activated protein kinase p38, whereby the Lck and Zap70 tyrosine kinases directly activate p38. Here we report that alternatively activated p38 associated with the Dlgh1 MAGUK scaffold protein. 'Knockdown' of Dlgh1 expression blocked TCR-induced activation of p38 and the transcription factor NFAT but not of the mitogen-activated protein kinase Jnk or transcription factor NF-κB. A Dlgh1 mutant incapable of binding p38 failed to activate NFAT. Along with reports that the CARMA1 MAGUK scaffold protein coordinates activation of Jnk and NF-κB but not of p38 or NFAT, our findings identify MAGUK scaffold proteins as 'orchestrators' of TCR signal specificity.
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Acknowledgements
We thank members of the Miceli lab for critical reading of the manuscript, and J. Ashwell for the antibody to p38 phosphorylated at Tyr323 and for critical reading of the manuscript. The plasmid encoding the NFAT-responsive luciferase reporter construct was a gift from K. Siminovitch (Samuel Lunenfeld Research Institute of Medicine). Supported by the US National Institutes of Health (RO1A1056155 to M.C.M.), the United States Public Health Service (GM07185 to J.L.R.), the University of California at Los Angeles Graduate Division (J.L.R.) and National Institutes of Allergy and Infectious Disease and National Institutes of Health (2-T32-AI-07323 to T.T. and AI07126-30 to L.A.H.). Flow cytometry was done at the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility, which is supported by the National Institutes of Health (CA-16042 and AI-28697).
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J.L.R. performed all of the experiments shown; L.A.H provided assistance with calcium flux assays and whole cell lysate p-p38 and anti-phosphotyrosine immunoblots; T.T. provided assistance with mouse work, P.M. provided assistance with phospho-Tyr323 p38 induction and immunoblotting, M.Z. performed anti-phosphotyrosine immunoblots, and M.C.M. supervised all experiments.
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Supplementary information
Supplementary Fig. 1
A model whereby MAGUK driven signalosomes coordinate specific activation of NFAT and NF-κB. (PDF 128 kb)
Supplementary Table 1
Primers used to generate GST-Dlgh1 truncations. (PDF 67 kb)
Supplementary Table 2
Primers used to generate Dlgh1 PDZ point mutations. (PDF 67 kb)
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Round, J., Humphries, L., Tomassian, T. et al. Scaffold protein Dlgh1 coordinates alternative p38 kinase activation, directing T cell receptor signals toward NFAT but not NF-κB transcription factors. Nat Immunol 8, 154–161 (2007). https://doi.org/10.1038/ni1422
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DOI: https://doi.org/10.1038/ni1422
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