Activation of Mitogen-activated Protein Kinase Kinase (MKK) 3 and MKK6 by Type I Interferons

David E. Levy,Beata Majchrzak,Sandeep Batra,Leonidas C. Platanias,Roger J. Davis,Yongzhong Li,Matthias Gaestel,Eleanor N. Fish,Antonella Sassano

doi:10.1074/jbc.m410972200

Abstract

There is accumulating evidence that the p38 MAP kinase pathway plays important roles in Type I interferon (IFN) signaling, but the mechanisms regulating p38 activation during engagement of the Type I IFN receptor remain to be defined. We sought to identify the events that lead to activation of the p38 MAP kinase in response to Type I IFNs. Our data demonstrate that treatment of sensitive cell lines with IFNalpha results in activation of both MAP kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6). Such IFN-inducible activation of MKK3 and MKK6 is essential for downstream phosphorylation and activation of the p38 MAP kinase, as shown by studies using mouse embryonic fibroblasts (MEFs) with targeted disruption of the Mkk3 and Mkk6 genes (MKK3-/- MKK6-/-). Similarly, IFN-dependent activation of the downstream effectors of p38, MAPKAPK-2 and MAPKAPK-3, is not detectable in cells lacking Mkk3 and Mkk6, demonstrating that the function of these MAP kinase kinases is required for full activation of the p38 pathway. To define the functional relevance of MKK3/6 engagement in Type I IFN signaling, IFN-inducible gene transcription was evaluated in the MKK3/MKK6 double knock-out cells. IFNalpha- and IFNbeta-dependent transcription via either interferon-stimulated response element or IFNgamma activated site elements was defective in MKK3 -/-/MKK6 -/- MEFs in luciferase reporter assays. In addition, IFN-dependent induction of two genes known to be of importance in the generation of IFN responses, Isg15 and Irf-9, was diminished in the absence of Mkk3 and Mkk6. The effects of Mkk3 and Mkk6 on IFN-dependent transcription were unrelated to any effects on the phosphorylation and activation of STAT proteins, indicating the presence of a STAT-independent mechanism. Altogether, our findings demonstrate that MKK3 and MKK6 are rapidly activated during engagement of the Type I IFN receptor and play important roles in Type I IFN signaling and the generation of IFN responses.

Highlights

Type I interferons (IFNs)1 are pleiotropic cytokines that mediate antitumor, antiviral, and immunomodulatory responses in vitro and in vivo [1,2,3,4,5,6,7,8]
We initially examined whether treatment of sensitive cell lines with IFN␣ results in phosphorylation of the mitogen-activated protein (MAP) kinase kinase 3 (MKK3) and/or MAP kinase kinase 6 (MKK6) kinases
KT-1 or Molt-4 cells were incubated for different times in the presence or absence of IFN␣, and cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody that recognizes the phosphorylated forms of MKK3 on serine 189 and/or MKK6 on serine 207

Summary

Introduction

Type I interferons (IFNs) are pleiotropic cytokines that mediate antitumor, antiviral, and immunomodulatory responses in vitro and in vivo [1,2,3,4,5,6,7,8]. In the present study we provide evidence that IFN␣ or IFN␤ treatment of sensitive cell lines results in activation of MAP kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6). Such activation is critical for activation of the p38 MAP kinase, as in cells with targeted disruption of both the Mkk and Mkk genes there is complete abrogation of the ability of Type I IFNs to induce p38 activation. IFN-inducible activation of MAPKapK-2 is MKK3/6-dependent and interferon stimulated gene (ISG) expression is defective in MAPKAPK-2Ϫ/Ϫ MEFs, indicating that this kinase acts as a downstream effector for the MKK3/6-p38 MAP kinase pathway, to mediate transcriptional regulation of ISGs

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Results

Conclusion