| Abstrakt: |
Protein kinase C-ϑ (PKCϑ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCϑ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCϑ is necessary and sufficient for PKCϑ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCϑ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCϑ-Thr335-to-Ala abolished the ability of PKCϑ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCϑ binding to Pin1, suggesting that Pin1-PKCϑ association is contingent upon the phosphorylation of the PKCϑ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCϑ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCϑ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCϑ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCϑ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCϑ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCϑ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCϑ, indicating the specificity of the Pin1-PKCϑ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCϑ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCϑ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCϑ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase. [ABSTRACT FROM AUTHOR] |
