The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C ? via a critical Phospho-Thr-Pro motif in the V3 regulatory domain

Protein kinase C-? (PKC?) 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 PKC? 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 PKC? is necessary and sufficient for PKC? IS localization and function. Herein, we highlight the importance of Thr(335)-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKC? and its subsequent IS localization. We demonstrate that the phospho-Thr(335)-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 PKC?-Thr(335)-to-Ala abolished the ability of PKC? to interact with Pin1, while Thr(335) replacement by a Glu phosphomimetic, restored PKC? binding to Pin1, suggesting that Pin1-PKC? association is contingent upon the phosphorylation of the PKC?-Thr(335)-Pro motif. Similarly, the Pin1 mutant, R(17)A, failed to associate with PKC?, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKC? interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKC? phospho-Thr(335)-Pro motif, to form a stable interaction between Pin1 and PKC?. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKC? complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKC?-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 PKC?, indicating the specificity of the Pin1-PKC? association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKC? and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA(307-319))-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKC? and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr(335)-Pro motif within the PKC?-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.

Automated summary

Protein kinase C-α (PKC-α) is selectively expressed in T lymphocytes and plays a crucial role in T cell activation and proliferation. The phosphorylation of Thr(335)-Pro residue in the regulatory domain of PKC-α is important for its activation and localization at the immunological synapse. The peptidyl-prolyl cis-trans isomerase Pin1 interacts with PKC-α through the phosphorylated Thr(335)-Pro motif, and this interaction is involved in early activation events in TCR-triggered T cells.