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Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.

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]

PKC[theta] plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-[kappa]B, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKC[theta] to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKC[theta], we addressed here the question of whether the translocation and activation of PKC[theta] in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLC[gamma]1-deficient T cell line, or a dominant negative PLC[gamma]1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKC[theta] are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKC[alpha]. Membrane or lipid raft recruitment of PKC[theta] (but not PKC[alpha]) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKC[theta], but did not associate with it. These results provide evidence that a nonconventional PI3-K- and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKC[theta] in T cells.