An Essential Role for Perforin-2 in Type I IFN Signaling [INNATE IMMUNITY AND INFLAMMATION]

Key Points

  • Type I IFN–mediated JAK–STAT signaling is impaired in Perforin-2–deficient cells.

  • Perforin-2 independently associates with IFNAR1 and IFNAR2 in resting cells.

  • Perforin-2 is critical for the activation of Jak1 and Tyk2 upon IFN-β stimulation.

Visual Abstract

Figure1

Abstract

Type I IFNs play a complex role in determining the fate of microbial pathogens and may also be deleterious to the host during bacterial and viral infections. Upon ligand binding, a receptor proximal complex consisting of IFN-α and -β receptors 1 and 2 (IFNAR1, IFNAR2, respectively), tyrosine kinase 2 (Tyk2), Jak1, and STAT2 are assembled and promote the phosphorylation of STAT1 and STAT2. However, how the IFNARs proximal complex is assembled upon binding to IFN is poorly understood. In this study, we show that the membrane-associated pore-forming protein Perforin-2 (P2) is critical for LPS-induced endotoxic shock in wild-type mice. Type I IFN–mediated JAK–STAT signaling is severely impaired, and activation of MAPKs and PI3K signaling pathways are delayed in P2-deficient mouse bone marrow–derived macrophages, mouse embryonic fibroblasts (MEFs), and human HeLa cells upon IFN stimulation. The P2 N-glycosylated extracellular membrane attack complex/perforin domain and the P2 domain independently associate with the extracellular regions of IFNAR1 and IFNAR2, respectively, in resting MEFs. In addition, the P2 cytoplasmic tail domain mediated the constitutive interaction between STAT2 and IFNAR2 in resting MEFs, an interaction that is dependent on the association of the extracellular regions of P2 and IFNAR2. Finally, the constitutive association of P2 with both receptors and STAT2 is critical for the receptor proximal complex assembly and reciprocal transphosphorylation of Jak1 and Tyk2 as well as the phosphorylation and activation of STAT1 and STAT2 upon IFN-β stimulation.

Footnotes

  • 1 R.M. and R.H. are co-first authors.

  • 2 E.R.P. deceased.

  • This work was supported by National Institutes of Health Grants CA039201, CA109094, AI0073234, and AI096396 (to E.R.P.); the Lois Pope Life Foundation Development Fellowship (to R.M.); and American Cancer Society Grant RSG-16-254-01-MPC (to N.S.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BMDM
    bone marrow–derived macrophage
    Co-IP
    coimmunoprecipitation
    CT
    cytoplasmic tail
    IFNAR
    IFN-α and -β receptor
    ISRE
    IFN-sensitive response element
    MACPF
    membrane attack complex/perforin
    MEF
    mouse embryonic fibroblast
    P2
    Perforin-2
    PBST
    PBS with 0.05% Tween 20
    Rap1
    Ras-related protein 1
    shRNA
    short hairpin RNA
    TM
    transmembrane
    TYK2
    tyrosine kinase 2
    VSV-GFP
    vesicular stomatitis virus–encoding GFP.
  • Received August 21, 2019.
  • Accepted February 12, 2020.

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