Characterization of TCR-induced phosphorylation of PKCtheta in primary murine lymphocytes

Protein kinase C theta (PKCtheta) is a member of the "novel" PKC subfamily which plays a critical role in T-cell activation. Following T-cell stimulation, PKCtheta translocates to the center of the immunological synapse where it co-localizes with the T-cell receptor (TCR). PKCtheta is required for the activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), which regulate the production of interleukin-2 (IL-2), a necessary step for the deployment of T-cell effector functions. By using primary murine T lymphocytes we have investigated regulatory phosphorylation events on PKCtheta which may impinge on its activity or its interaction with other signaling mediators. Here, we describe a TCR stimulation-induced Ser/Thr phosphorylation event on PKCtheta that takes place simultaneously with its recruitment to cellular membranes and which can be detected as an electrophoretic mobility shift. By analyzing Ser and Thr point mutants, we find that phosphorylation of Ser-695 in the hydrophobic motif is one, but not the only residue involved in the mobility shift. Interestingly, Ser-695 appears to be required to trigger further phosphorylation events at adjacent Thr-692 and Thr-703 residues, which also participate in the mobility shift. We show that phosphorylation at these residues is not due to auto-phosphorylation, but requires instead Src family kinase and phosphatidylinositol 3-kinase (PI3K) activities. We further show that activation-induced phosphorylation of PKCtheta correlates with its function but not with its kinase activity, suggesting that phosphorylation of PKCtheta plays a role in its interaction with upstream or downstream effectors.