Conventional Protein Kinase C-α (PKC-α) and PKC-β Negatively Regulate RIG-I Antiviral Signal Transduction

Retinoic acid-inducible gene I (RIG-I) is a key sensor for viral RNA in the cytosol, and it initiates a signaling cascade that leads to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent signaling function is regulated by the dynamic balance between phosphorylation and TRIM25-induced K63-linked ubiquitination. While ubiquitination of RIG-I is critical for RIG-I's ability to induce an antiviral IFN response, phosphorylation of RIG-I at S(8) or T(170) suppresses RIG-I signal-transducing activity under normal conditions. Here, we not only further define the roles of S(8) and T(170) phosphorylation for controlling RIG-I activity but also identify conventional protein kinase C-alpha (PKC-alpha) and PKC-alpha as important negative regulators of the RIG-I signaling pathway. Mutational analysis indicated that while the phosphorylation of S(8) or T(170) potently inhibits RIG-I downstream signaling, the dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression, gene silencing, and specific inhibitor treatment demonstrated that PKC-alpha/beta are the primary kinases responsible for RIG-I S(8) and T(170) phosphorylation. Coimmunoprecipitation showed that PKC-alpha/beta interact with RIG-I under normal conditions, leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination, and thereby RIG-I-mediated IFN induction. PKC-alpha/beta double-knockdown cells exhibited markedly decreased S(8)/T(170) phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-alpha/beta-induced RIG-I phosphorylation is a critical regulatory mechanism for controlling RIG-I antiviral signal transduction under normal conditions.