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

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 (PKCθ^HR2A), we showed that pTyr binding by PKCθ was required for TCR-induced T cell activation, proliferation, and T_H2 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. PKCθ^HR2A-expressing CD4⁺ T cells displayed defects not only in known PKCθ-dependent signaling events, such as nuclear factor κB (NF-κB) activation and T_H2 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.