Research ArticleImmunology

Phosphotyrosine-dependent interaction between the kinases PKCθ and Zap70 promotes proximal TCR signaling

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Sci. Signal.  16 Apr 2019:
Vol. 12, Issue 577, eaar3349
DOI: 10.1126/scisignal.aar3349

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PKCθ zaps the immunological synapse

Protein kinase C-θ (PKCθ) translocates to the center of the immunological synapse in antigen-stimulated T cells and plays a role in their activation. Unlike other PKC isoforms, PKCθ and its closest relative PKCδ bind to phosphotyrosines through their N-terminal C2 domains. Xie et al. found that phosphotyrosine binding by the PKCθ C2 domain was required for T cell activation, proliferation, and TH2 cell differentiation. The C2 domain interacted with the phosphorylated form of the kinase Zap70, which is critical for signaling downstream of the T cell receptor (TCR). Phosphotyrosine binding by PKCθ was required for its recruitment to the immunological synapse and for Zap70-dependent TCR signaling. Given limited success in developing clinically useful, highly specific catalytic inhibitors of PKCθ or Zap70, targeting the PKCθ-Zap70 interaction may be an alternative strategy for developing selective immunosuppressive drugs.

Abstract

Protein kinase C-θ (PKCθ) is an important component of proximal T cell receptor (TCR) signaling. We previously identified the amino-terminal C2 domain of PKCθ as a phosphotyrosine (pTyr)–binding domain. Using a mutant form of PKCθ that cannot bind pTyr (PKCθHR2A), we showed that pTyr binding by PKCθ was required for TCR-induced T cell activation, proliferation, and TH2 cell differentiation but not for T cell development. Using tandem mass spectrometry and coimmunoprecipitation, we identified the kinase ζ-associated protein kinase of 70 kDa (Zap70) as a binding partner of the PKCθ pTyr-binding pocket. Tyr126 of Zap70 directly bound to PKCθ, and the interdomain B residues Tyr315 and Tyr319 were indirectly required for binding to PKCθ, reflecting their role in promoting the open conformation of Zap70. PKCθHR2A-expressing CD4+ T cells displayed defects not only in known PKCθ-dependent signaling events, such as nuclear factor κB (NF-κB) activation and TH2 cell differentiation, but also in full activation of Zap70 itself and in the activating phosphorylation of linker of activation of T cells (LAT) and phospholipase C-γ1 (PLCγ1), signaling proteins that are traditionally considered to be activated independently of PKC. These findings demonstrate that PKCθ plays an important role in a positive feedback regulatory loop that modulates TCR-proximal signaling and, moreover, provide a mechanistic explanation for earlier reports that documented an important role for PKCθ in T cell Ca2+ signaling. This PKCθ-Zap70 interaction could potentially serve as a promising and highly selective immunosuppressive drug target in autoimmunity and organ transplantation.

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