Loss-of-function phenotype of a PKCθ T219A knockin mouse strain

Cell Commun Signal. 2019 Nov 6;17(1):141. doi: 10.1186/s12964-019-0466-8.

Abstract

Background: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line.

Methods: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry.

Results: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression.

Conclusion: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.

Keywords: Interleukin 2 (IL-2) production; NF-κB; NFAT; Protein kinase C θ (PKC θ); T cell activation; Thr-219 autophosphorylation site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cytokines / metabolism
  • Gene Knock-In Techniques*
  • Mice
  • Phenotype*
  • Protein Kinase C-theta / genetics*
  • Protein Kinase C-theta / metabolism*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / metabolism

Substances

  • Cytokines
  • Protein Kinase C-theta