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

• Published in: Frontiers in immunology Vol. 14; p. 1126464
• Main Authors: Anto, Nikhil Ponnoor, Muraleedharan, Amitha, Nath, Pulak Ranjan, Sun, Zuoming, Keasar, Chen, Livneh, Etta, Braiman, Alex, Altman, Amnon, Kong, Kok-Fai, Isakov, Noah
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 2023
• Subjects: Animals; Humans; immunological synapse; Immunology; Mice; Mice, Inbred C57BL; NIMA-Interacting Peptidylprolyl Isomerase - genetics; Peptides; Peptidylprolyl Isomerase - chemistry; Peptidylprolyl Isomerase - genetics; Peptidylprolyl Isomerase - metabolism; Pin1; PKCθ; Proline - chemistry; Proline - metabolism; prolyl isomerase; protein kinase; Protein Kinase C-theta - genetics; Receptors, Antigen, T-Cell; T cell activation; immunological synapse; Pin1; prolyl isomerase; T cell activation; PKCθ; protein kinase
• ISSN: 1664-3224; 1664-3224
• DOI: 10.3389/fimmu.2023.1126464

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 Thr -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-Thr -Pro motif serves as a putative binding site for the peptidyl-prolyl isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr -to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr -Pro motif. Similarly, the Pin1 mutant, R A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr -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 (HA )-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 Thr -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 isomerase.