The Peptidyl-Prolyl Isomerase Pin1 Negatively Regulates Protein Kinase C-θ in Activated T Cells

Introduction

Protein kinase C-θ (PKCθ) is highly expressed in T lymphocytes where it regulates essential functions required for cell activation and proliferation. We observed that PKCθ possesses Ser/Thr-Pro motifs that, upon phosphorylation, might serve as putative binding sites for the Pin1 enzyme, which catalyzes cis-trans isomerization of peptide bonds at phospho-Ser/Thr-Pro motifs. In view of the emerging importance of PKCθ in T cell biology, the present study focuses on the Pin1-dependent regulation of PKCθ in TCR-activated T cells.

Materials and Methods

Cell lines used in this study include sublines of Jurkat, a human leukemia T cell lines, and C57BL/6J mouse spleen and thymus cells. Protein binding studies were performed by immunoprecipitation and immunoblotting, using PKCθ and Pin1 specific Abs, and pull-down assays, using bead-immobilized GST-Pin1 fusion proteins. Intracellular localization of Pin1 and PKCθ was determined by immunofluorescence studies and confocal microscopy. The effect of Pin1 on the catalytic activity of PKCθ was tested in vitro in a kinase assay, using γ-³²P-ATP and myelin basic protein, as a substrate.

Results and Discussion

Coimmunoprecipitation studies performed using Jurkat T cells, mouse splenic T cells and thymocytes revealed the association between PKCθ and Pin1. Binding of Pin1 to PKCθ was augmented following TCR crosslinking, and downregulated when performed in the presence of an active phosphatase. Our studies demonstrated Thr³³⁵-Pro-Gly motif within the V3 region in the regulatory domain of PKCθ is the site of Pin1 binding, as the mutagenesis of Thr³³⁵ to Ala (T/A) which completely abolished the ability of PKCθ to bind Pin1. Using bead-immobilized GST fusion proteins in a pull-down assay, we found that PKCθ binding is mediated by the Pin1 N-terminal WW domain. Immunofluorescence studies demonstrated that TCR crosslinking induces partial colocalization of Pin1 and PKCθ at the cell membrane. Furthermore, in vitro kinase assay indicated that human recombinant Pin1 inhibits the ability of Jurkat T cell- and mouse splenic T cell-derived PKCθ to phosphorylate myelin basic protein. Inclusion of juglone, a Pin1 inhibitor reversed the effect of Pin1 to inhibit PKCθ catalytic activity in the in vitro kinase assay, suggesting that Pin1 mediates its effect by isomerization of PKCθ.

Conclusions

Pin1 is a PKCθ-binding partner in T cells. Its association with PKCθ increases following TCR stimulation and in vitro studies suggest that it functions as a negative regulator of PKCθ activity.