Mouse monoclonal antibodies (mAbs) directed against PKCθ (#610090), and hamster mAbs directed against CD3ϵ (145-2C11, #550275) were from BD Transduction Laboratories (Lexington, KY). Hamster mAbs directed against mouse CD28 (clone 37.51; #102102) were from Biolegend (San Diego, CA). Rabbit anti-Pin1 (#76309) mAbs and rabbit anti-FK506 binding protein 12 (FKBP12) (#92459) mAbs, rabbit anti-IgG isotype control mAbs (#172730) and goat polyclonal anti-mouse IgG (#6708) Abs, Goat anti-hamster IgG (#5738) Abs and CytoPainter Cell Staining Reagent were from Abcam (Cambridge, UK). Rabbit anti-Cyclophilin A mAb (CypA; #133494) was from Santa Cruz Biotechnology, Inc. Horseradish peroxidase (HRP)-conjugated sheep anti-mouse, donkey anti-rabbit and Goat-anti-hamster immunoglobulin Abs, ECL (Electrochemiluminescence), glutathione-coupled sepharose beads, protein A-, G-, and A/G-agarose beads, and nitrocellulose membranes were from Amersham Pharmacia Biotech (Uppsala, Sweden). Mouse anti-β actin (clone AC-15) mAb were from Sigma-Aldrich. Mouse anti-human CD3ϵ (OKT3) and anti-c-Myc mAbs (clone 9E10) were prepared as previously described (19). Protease inhibitor cocktail tablets were from Roche (Basel, Switzerland). Mouse anti-Xpress (#R910-25), Alexa Fluor™ 488-, 546- and 633-conjugated rabbit and mouse Ig Abs, goat anti-mouse Ig Abs, and Alexa Fluor™ 633-conjugated phalloidin were from Thermo Scientific, Inc. (Waltham, MA). Molecular weight marker (Page Ruler) was from Thermo Fisher Scientific. Tricine sample buffer (#161-0739) was from Bio-Rad Laboratories, Inc. Ampicillin was from AppliChem Inc. (Maryland Heights, MO). Chemically competent DH5α™ cells were from Invitrogen (Carlsbad, CA). Calf intestinal alkaline phosphatase (CIP) and lambda phosphatase were from New England Biolabs, Inc. (Ipswich, MA).

Spleen and thymus cells were obtained from 6-8-wk-old C57BL/6J female mice that were housed under controlled conditions with 12 h light/dark cycle and free access to food and water. Splenic and thymic T cells were obtained by density gradient centrifugation using Ficoll-Paque™ solution (Thermo Fisher Scientific). Wild-type C57BL/6 mice were purchased from Harlan. This study was approved in advance by the Ben-Gurion University Institutional Animal Care and Use Committee and conducted in accordance with the Israeli Animal Welfare Act following the guidelines of the Guide for Care and Use of Laboratory Animals (National Research Council 1996). The Animal ethical clearance protocol used for this research is IL-44-08-2017.

Cell lines used in this study include the human T cell leukemia, Jurkat (clone E6-1; ATCC^® TIB-152™), Jurkat Tag, which stably expresses the Simian Vacuolating Virus 40 (SV40)-derived large T Ag, TCR-deficient Jurkat T cell line, CH7C17, which was transfected with the influenza hemagglutinin (HA^307-319) peptide-specific, DR1-restricted HA1.7 TCR, and the EBV-transformed human B cell line, LG2, which expresses the HLA-DR1 and B7.1 surface receptors, human Burkitt’s lymphoma line, Raji, and African green monkey kidney-derived COS-7 cells. Jurkat cell lines were maintained at a logarithmic growth phase in RPMI (Sigma), supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin (all from Biological Industries, Kibbutz Beit Haemek, Israel)) (complete RPMI), and grown in 75-cm² tissue culture flasks (CellStar, Greiner, Germany). COS-7 cells were maintained at a logarithmic growth phase in DMEM (Sigma) supplemented with 10% heat-inactivated FCS, 50 units/ml penicillin, and 50 μg/ml streptomycin (complete DMEM) in 145 mm tissue culture plates (CellStar, Greiner, Germany).

Jurkat T cell stimulation via the TCR was performed by incubation with anti-CD3 mAbs (OKT3, 30 min on ice) plus cross-linking with a secondary Ab, goat anti-mouse IgG, for the indicated time intervals, at 37°C. TCR stimulation of mouse spleen and thymus cells was carried out using hamster anti-CD3ϵ mAbs (145-2C11, 30 min on ice) plus cross-linking with a secondary Ab, goat anti-hamster IgG, for the indicated time intervals, at 37°C.

pGEX bacterial expression vectors encoding GST-Pin1, GST-Pin1-C113A, and GST-Pin1-R17A were gifts of Hung-Ying Kao (Case Western Reserve University). Luciferase reporter constructs for NFAT, NF-κB, CD28 response element RE/AP, and the β-Galactosidase (β-Gal) reporter plasmids were obtained from M. Karin. Empty vector or expression vectors for Xpress-tagged wild-type [WT (T³³⁵P)], or mutant PKCθ (ΔV1, ΔV3, ΔV4, and ΔV6) were prepared as previously described (9).

Myc-tagged peptides, KS21 and pKS21 peptides, which include the PKCθ-V3 domain-derived Thr³³⁵-Pro motif plus ten amino acids on either side of this sequence were from GL Biochem (Shanghai) Ltd. The KS21–Myc peptide (KNEARPPCLPTPGKREPQGIS-EQKLISEEDL) lacks a phosphoryl group on Thr³³⁵ while pKS21-Myc (KNEARPPCLP(pT)PGKREPQGIS-EQKLISEEDL) includes a phosphoryl group on Thr³³⁵.

PKCθ mutants [T/A, A³³⁵P; T/E, E³³⁵P] were generated by site-directed mutagenesis and overlapping PCR.

Non-adherent cells: Jurkat T cells maintained at a logarithmic phase were washed in supplement-free RPMI 1640, resuspended either in 700 μl transfection buffer (1.8 g/ml sucrose, 2nM DTT in RPMI 1640) or in 250 μl Mirus Ingenio^® electroporation solution and transferred into 0.4 cm-gap Gene Pulser cuvettes (BioRad) (20x10⁶ cell/700 μl/cuvette). Plasmid DNA (10 μg/group, unless otherwise indicated) was added to the cuvettes, and electroporation was performed using a BioRad Gene Pulser (250V, 950mF). The cells were then cultured in 50 ml of complete RPMI 1640 in 145 mm tissue culture plates for 48 h. Unless otherwise indicated, all nucleofection experiments were carried out in triplicates using 3 separate dishes for each point.

Adherent cells: COS-7 cells maintained at a logarithmic phase were collected from culture dishes by trypsin treatment, washed in supplement-free DMEM, resuspended in 5 ml of 10% FCS-containing DMEM (2x10⁶ cells/ml), and cultured in CellStar tissue culture flasks (Greiner Bio-one 690170). Within 24 hours of plating, adherent cells were transfected with the indicated DNA (5μg/group, unless otherwise indicated) using the PEI (Polysciences) transfection reagent in a 1:3 (DNA: PEI) ratio. The cells were then cultured in 10 ml complete DMEM in 75 cm² tissue culture flasks for 48h.

Cell lysates were prepared by resuspension of cells in a lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na₃VO₄, protease inhibitor cocktail (1 tab/10 ml), and 1% Triton X-100), followed by a 30 min incubation on ice. Lysates were centrifuged at 13,000xg for 30 min at 4°C and the nuclear-free supernatants were used for immunoprecipitation. Immunoprecipitation was performed by pre-adsorption of primary Abs onto protein A-, G-, or protein A/G-coupled beads, based on the differential affinities of the proteins to specific Abs, for 2 h at 4°C. Equal volumes of 2x Laemmli Sample Buffer were added to immunoprecipitates or whole-cell lysates, which were vortexed, boiled for 5 min, and fractionated by SDS-PAGE, either on 10%, or 12.5% polyacrylamide gels, and 16.5% Tris-Tricine gels, using Bio-Rad Mini-PROTEAN II cells. Proteins from the gel were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) at 100V for 1 h, using BioRad Mini Trans-Blot transfer cells. After 1 h of blocking with 3% BSA in TBST at 37°C, the nitrocellulose membranes were incubated in the presence of the indicated primary Abs, followed by incubation with HRP-conjugated secondary Abs. Immunoreactive protein bands were visualized using an ECL reagent and autoradiography.

pGEX bacterial expression vectors were used to transform Escherichia Coli DH5α™ competent cells and GST-fusion proteins were prepared as described (20). Pull-down assays were performed by incubation of bead-immobilized GST or GST fusion proteins (2-5 μg/sample) with cell lysates at 4°C on a rotator for 4 h. The beads were washed (x3) in lysis buffer and bound proteins were eluted and subjected to SDS-PAGE under reducing conditions, followed by immunoblotting.

Thymi from 6-8-wk-old C57BL/6J mice were immersed in a 4% paraformaldehyde solution at 4°C overnight and embedded into paraffin blocks that were cut into 4-6 µm slices. The tissue sections were mounted on positively-charged glass slides and dried for 1 h at 60°C, deparaffinized in xylene, and rehydrated in absolute ethanol. The slides underwent an antigen retrieval step in sodium citrate buffer (pH 6) in a steam pot. After boiling, sections were kept for cooling at room temperature for 30 min.

Jurkat T cells (2×10⁶/group) were seeded on poly-L-lysine-coated 8 well µ-slide (ibidi Ltd.) and deposited on the slides by centrifugation at 1200 rpm for 5 min. Cells were fixed in 4% paraformaldehyde (Sigma)/PBS for 5 min at room temperature, permeabilized with PBS/0.1% Triton X-100 for 5 min, and blocked with Ab Diluent blocking solution (GBI Labs) for 1 h at RT. Cells were then incubated with mouse anti-PKCθ mAb and rabbit anti-Pin1 mAb in blocking buffer for 1 h at room temperature. After three washes in PBS, the cells were incubated with Alexa Fluor 546-conjugated anti-mouse (or rabbit) Ig and Alexa Fluor 488-conjugated anti-rabbit (or mouse) Ig secondary Abs for 1 h in the dark at RT and counterstained with DAPI (1µg/ml) in PBS for 15 min. Staining specificity was tested by parallel cell treatment with the same reagents, except for the primary Abs.

Thymus sections were incubated with 3% normal goat serum (1 h), followed by incubation with goat anti-mouse IgG (1 h in a humidified chamber at room temperature) to block nonspecific binding. After careful removal of excess serum, the sections were overlaid with mouse anti-PKCθ mAbs and rabbit anti-Pin1 mAbs for 16 h in a humidified chamber at 4°C. The sections were washed in PBS and incubated with secondary Abs plus DAPI, as above. A control experiment was performed by staining of C57BL/6J mouse thymic sections with labeled Alexa Fluor™546-conjugated goat anti-mouse and Alexa Fluor™488-conjugated goat anti-rabbit secondary Abs, in order to rule out the possible basal, and non-specific staining. The coverslips were mounted on slides using DAKO mounting medium and imaged by Olympus FluoView FV1000 laser-scanning confocal microscope. Immunofluorescence images represent single 2-D confocal sections. The extent of colocalization of Pin1 and PKCθ was quantified using the JACoP ImageJ plugin (21).

Immunological synapse studies were performed as previously described (22). Jurkat E6.1 T cells transfected with an expression vector for Xpress-tagged wild-type or mutant PKCθ, were incubated at a ratio of 1:1 with Raji B cells pre-pulsed with Staphylococcus enterotoxin E (SEE) for 5 min. After fixation, conjugates were stained with mouse anti-Xpress plus a secondary Alexa fluor 488-coupled Ab (AF488) and counterstained with DAPI. Similarly, Jurkat-CH7C17 T cells, which express the influenza hemagglutinin (HA^307-319) specific TCR, were incubated with peptide-loaded antigen-presenting cells (APCs), LG2, at a ratio of 1:2 at 37°C for 5 min. APCs were pre-loaded with 200 μg/ml of HA^307-319 peptide, or an inactive HA peptide, in which Lys316 was replaced by Glu (K316E), for 3 hours at 37°C. LG2 cells were prestained using CytoPainter Cell Staining Reagent prior to the cell mixing. Following 5 min of co-incubation, the cells were fixed, permeabilized, and analyzed by confocal microscopy. Immunofluorescence staining was performed using the primary Abs: mouse anti-PKCθ and rabbit anti-Pin1, followed by incubation with Alexa Fluor 633-conjugated phalloidin and: Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 546-conjugated anti-mouse IgG. CytoPainter was detected at 405nm (Olympus FluoView FV1000 laser-scanning confocal microscope).

Using the Swiss-PdbViewer package (23), we modeled the complex of the WW domain of Pin1 with PKCθ using the structure of another Pin1 complex as a template. The crystal structure of Pin1 bound to a phosphorylated peptide, via its WW domain, was solved by Verdecia et al. (PDB code 1F9A) (24). The peptide in that structure, which is derived from RNA polymerase II, is Tyr-pSer-Pro-Thr-pSer¹⁷⁴-Pro-Ser. While the crystal structure also includes the catalytic domain of Pin1, the domain interface is rather small, and does not include the peptide. Thus, our modeling includes only the WW domain of Pin1. We threaded the Pro-Leu-Pro-Thr³³⁵-Pro-Gly peptide of PKCθ on the bound peptide, with pSer¹⁷⁴-Pro and pThr³³⁵-Pro respectively as anchors. The major binding pocket in the crystal structure includes Ser¹⁶, Arg¹⁷, and Ser¹⁸ that coordinate the phosphate group and Trp³⁴ and Tyr²³ that line a hydrophobic pocket that fit the proline side chain. The corresponding PKCθ moieties fit this pocket in the model by construction, as they adopt the coordinates of almost identical residues. Tyr²³ also lines a minor binding pocket, together with Phe²⁵. In the crystal structure this pocket is occupied by a proline side chain, in the model it snugly fit a Leucine side chain, after adjustment of side-chain rotamers. All the other peptide residues hardly contact the WW domain, and thus replacing them with by the PKCθ residues in the model does not confer any clashes or incompatibility.

Plasmids for silencing Pin1 gene, pSuper-retro-puro-shPin1, and a control vector, pSuper-retro-puro-shControl were gifts of Prof. Akihide Ryo, Yokohama City University: (pSUPER RNAi System, Oligoengine). A mix containing a packaging plasmid, pPAX2 (900 ng), an envelope plasmid pVSV-G (100 ng), and the selected pSuper-retro-puro-shPin1 or pSuper-retro-puro-shControl (1 μg) was prepared in OPTI-MEM serum-free media (Invitrogen, #31985-070) The plasmid mix was added to an LT mix containing Transit IT^®-LT1 transfection reagent and Opti-MEM. The transfection mix was added to the HEK293T packaging cells, which were maintained in DMEM containing 10% FBS and 1% Pen-Strep. On the next day, the medium was replaced by adding complete fresh DMEM medium. After an additional day, the medium was collected and spun at 1,250 rpm for 5 minutes. Polybrene (8µg/ml) was added to the medium, which was then filtered through a 0.44µm filter and used for transfection of Jurkat cells. The Jurkat cells (2x10⁵) were incubated with the transfection medium for 5 hours, and Pin1 gene-silenced cells were obtained by cell growth for 48 hours in a selection medium containing various concentrations of puromycin.

Statistical analysis was carried out using GraphPad Prism software Version 9.2.0. Microscopy data were quantified using the ImageJ plugin, JACoP. Statistical significance of differences between groups of averaged data points was assessed using the One-way ANOVA (Tukey’s Multiple Comparison test) or Two-way ANOVA as indicated.

PKCθ possesses an N-terminal regulatory region with several conserved functional domains and a C-terminal catalytic region. A proline-rich (PR) motif within the V3 region in the PKCθ regulatory domain was recently found to be necessary and sufficient for PKCθ IS localization and function (9). Moreover, human PKCθ harbors several Ser/Thr-Pro motifs that are predicted to be phosphorable, posing them as additional potential regulatory sites. Among them, Thr³³⁵-Pro was of particular interest, owing to its location within the unique PR hinge region of PKCθ ( Figures 1A–C ). To test this conjecture, mutagenesis of this key Thr³³⁵ residue, followed by PKCθ-related functional aspects has been employed. Luciferase reporter assay and coimmunoprecipitation studies were utilized to test whether Thr³³⁵ plays a role in PKCθ activation and/or ability to interact with binding partners. Jurkat T cells were utilized for both assays following their nucleofection with wild-type or the Thr³³⁵ point mutants [T/A, A³³⁵P; T/E, E³³⁵P]. For the reporter assay, the cells were cotransfected with a luciferase reporter for PKCθ-downstream effector molecules, including NFAT and NF-κB, or the CD28 response element RE/AP, and tested following TCR crosslinking using anti-CD3/CD28 mAbs. For the binding assay, PKCθ wild-type or the Thr³³⁵ mutant-overexpressing Jurkat T cells were lysed and subjected for coimmunoprecipitation using anti-CD28 mAbs. Our results demonstrated that phosphorylation of Thr³³⁵ is required for the activation of PKCθ ( Figures 1D–G ) and its binding to the costimulatory receptor, CD28 ( Figure 1H ). Thr³³⁵-to-Ala amino acid substitution completely abolished the ability of PKCθ to be triggered or to bind CD28, while replacement of Thr³³⁵ by Glu, which mimics the approximate size and charge of phospho-Thr, restored these functions. Furthermore, a conjugate formation assay, utilizing Jurkat T cells, Raji B cells as APCs, and staphylococcal enterotoxin (SEE) as a TCR stimulant, followed by fluorescent cell staining and confocal microscopy confirmed that phospho-Thr³³⁵ is required for PKCθ localization at the IS ( Figures 1I, J ). These data highlight the potential role of the Thr³³⁵-Pro motif in the spatiotemporal regulation of PKCθ and suggest that regulation via this motif is phosphorylation-dependent.

Based on our preliminary observations, we hypothesized that the phosphorylation of Thr³³⁵-Pro would turn it into a presumed binding site for the Pin1 enzyme, which specifically recognizes phospho-S/T-P motifs. To determine individual domains of PKCθ that can bind Pin1, we used a heterologous cell system involving COS-7 cells transfected with different Xpress-tagged PKCθ truncation mutant vectors ( Figure 2A ) and tested their ability to coimmunoprecipitate Pin1. Following overexpression of the constructs listed in Figure 2A , an anti-Xpress mAb was used for protein immunoprecipitation from the lysates of the transfected COS-7 cells, followed by immunoblot with Pin1-specific mAbs. This experiment unraveled an important observation that the integrity of V3 region is essential for PKCθ in its ability to interact with the Pin1 protein ( Figure 2B ).

PKCθ is subjected to post-translational modifications, primarily by phosphorylation of multiple sites (25). To study whether Pin1 interaction with PKCθ is dependent on phosphorylation, Pin1 was immunoprecipitated from Jurkat T cell lysates and the immunoprecipitate was subjected to treatment with and without enzymatically active phosphatases, followed by gel electrophoresis and immunoblotting. We found that PKCθ that coimmunoprecipitated with anti-Pin1 mAb almost completely dissociated from PKCθ following the treatment with calf intestinal phosphatase (CIP) ( Figure 2C ) or λ protein phosphatase (λPP) ( Figure S1A ). The results indicate that interaction between the two proteins is phosphorylation-dependent. Finally, by utilizing an appropriate isotype control, we validated the specificity of PKCθ coimmunoprecipitation with Pin1 ( Figure S1B ).

Pin1 has a modular domain architecture comprising an N-terminal WW domain (amino acids 6-37), a flexible linker (amino acids 38-53), and a C-terminal parvulin-type catalytic PPIase domain (amino acids 54-163) ( Figure 3A ). The WW domain is responsible for Pin1’s binding to pSer/Thr-Pro motifs in substrate proteins, while the PPIase domain catalyzes the interconversion of the cis and trans isomers of proline amide bonds. Both domains recognize pS/T-P motifs (26). A substitution mutation, R¹⁷A, in the WW domain of Pin1 abolishes the ability of Pin1 to interact with its known substrates, whereas a substitution mutation, C¹¹³A, downregulates the Pin1 catalytic activity. To determine the Pin1 domain that interacts with PKCθ we used bead-immobilized GST fusion proteins that include Pin1 without or with substitution replacement in the WW (R¹⁷A) or catalytic (C¹¹³A) domains ( Figure 3B ). Using a lysate of TCR-stimulated Jurkat T cells in a pull-down assay we found that mutation in R17, but not in C113, abolished the ability of Pin1 to bind PKCθ ( Figure 3C ). The data indicate that the integrity of the WW domain, but not the PPIase domain, is essential for Pin1-PKCθ interaction in T cells.

Since we showed that PKCθ-V3 is required for Pin1 binding ( Figure 1 ), we extended our studies on whether the Thr³³⁵-Pro motif within PKCθ-V3 could be integral for the binding of Pin1. Hence, we conducted a study in COS-7 cells that were transfected with PKCθ WT, and mutants T³³⁵A, and T³³⁵E, immunoprecipitated the transiently expressed PKCθ from their lysates (using anti-Xpress mAbs) and tested for Pin1 coimmunoprecipitation. As expected, Pin1 coimmunoprecipitated with PKCθ from the lysate of COS-7 cells transfected with PKCθ WT. However, replacement of Thr³³⁵ by Ala completely abolished the ability of PKCθ to bind Pin1 whereas substitution of Thr³³⁵ by the Glu phosphomimetic, reinstated Pin1 binding ( Figure 3D ). The data shows that phosphorylation of the Thr³³⁵-Pro motif in the PKCθ-V3 is critical for Pin1 binding. We put forth an alternate approach to corroborate our observations by using two Myc-tagged 21-mer peptides, KS21, and pKS21, which correspond to a PKCθ-V3-derived amino acid sequence that includes Thr³³⁵ plus 10 adjacent amino acids on each side. While both peptides had identical sequences, only pKS21-Myc was phosphorylated on Thr³³⁵. When each of the two peptides was added to a lysate of human Jurkat T cells, both peptides were detected following immunoprecipitation with anti-Myc mAbs. However, coimmunoprecipitation of Pin1 occurred only in the presence of pKS21-Myc, corroborating the requirement for phosphorylation of PKCθ-V3 on the Thr³³⁵-Pro motif to turn PKCθ into a Pin1 binding partner ( Figure 3E ). In addition, in silico structural modeling analysis revealed a suggestive interaction between PKCθ-Thr³³⁵-Pro and the critical amino acid residues in the Pin1-WW binding domain (Arg¹⁷, Tyr²³, and Trp³⁴) to materialize a PKCθ-Pin1 complex ( Figure 3F and Figure SV1 ). Overall, these results demonstrate that Pin1-PKCθ interaction is mediated by the N-terminal WW domain of Pin1 and the PKCθ-pThr³³⁵-Pro motif.

To further analyze the ability of Pin1 to interact with PKCθ in T cells, we used Jurkat T cells and mouse primary cells derived ex vivo from C57BL/6J mouse thymus and spleen. The cells were stimulated by crosslinking their TCRs using OKT3 (Jurkat T cells) and 2C11 (splenic/thymic cells) mAbs and the lysates were subjected to Pin1 immunoprecipitation and tested for PKCθ coimmunoprecipitation. We found that PKCθ coimmunoprecipitated with Pin1 from TCR/CD3-stimulated splenic cells ( Figures 4A, B ) and Jurkat cells ( Figure S2A ) with a maximal association occurring at ~30 secs post-TCR stimulation. In contrast, in thymic cells, PKCθ interacted with Pin1 at basal level at all time points post-TCR-crosslinking ( Figure 4C ). The results show that association of Pin1 with PKCθ in TCR-triggered splenic T cells and Jurkat T cells occurs in a time kinetic which corresponds to that of the recruitment of PKCθ to the IS. The constitutive binding of Pin1 to PKCθ that was observed in thymocytes likely represents the ongoing exposure of a large proportions of the cells to signals leading to proliferation, expansion, differentiation and apoptosis during the negative and positive thymic selection processes. Pin1-PKCθ interaction was further validated by demonstrating the ability of GST-Pin1 to pull down PKCθ from TCR-stimulated Jurkat T cells ( Figure 4D ). We also analyzed the selectivity of the Pin1 PPIase to PKCθ by comparing the ability of other PPIases (CypA and FKBP12) to associate with it. Indeed, we found that Jurkat T cell-derived PKCθ selectively associates with Pin1 but not with other peptidyl-prolyl cis-trans isomerases ( Figure S2B ). Finally, we found that Pin1-PKCθ association can also be demonstrated using a reciprocal immunoprecipitation, where anti-PKCθ mAbs coimmunoprecipitate Pin1 from lysates of OKT3-stimulated Jurkat cells ( Figure 4E ).

PKCθ reside in the cytosol of resting T cells and translocate to the cell membrane upon activation (2) while majority of the Pin1 proteins in resting T cells are confined to the cell nucleus (27, 28). To define the subcellular location of PKCθ-bound Pin1 in TCR-activated T cells, mouse spleen and Jurkat T cells were stimulated with anti-CD3 (2C11/OKT3) mAbs for 30 seconds, followed by staining with fluorescently-labeled Pin1- and PKCθ-specific Abs. Cell analysis using confocal microscopy demonstrated an augmented association between Pin1 and PKCθ at the cell membrane ( Figure 5A , and Figure S3A ). Previous observations showing a constitutive association between Pin1 and PKCθ in mouse thymocytes prompted us to analyze the distribution of the same by staining of C57BL/6J thymic sections. Immunofluorescence staining and imaging analysis revealed a strong association between Pin1 and PKCθ at the membrane of thymocytes in the tissue section ( Figure 5B ). Control experiments, utilizing a fluorescently-labeled mouse, and rabbit secondary Abs, excluded the possibility of a basal level or non-specific staining of Pin1 and PKCθ ( Figure S3B ). The results corroborate the interaction of Pin1 and PKCθ at the membrane of T cells and hint for a possible involvement of Pin1 in the regulation of PKCθ and PKCθ-dependent early activation events in TCR-engaged T cells.

Since engagement of the TCR promotes PKCθ translocation to the center of the IS, we further analyzed whether the same applies for the PKCθ-bound Pin1 protein. We used Jurkat T cells (clone CH7C17), which express the influenza hemagglutinin (HA) peptide-specific Vβ3 TCR, and co-cultured them with antigen-fed LG2 B cells, as APC. Cell staining with fluorescently-labeled Pin1- and PKCθ-specific Abs and analysis using a confocal microscope we observed that in a fraction of antigen (HA peptide 307-319) -pulsed LG2 cells that formed conjugates with the CH7C17 T cells, Pin1 and PKCθ colocalized at the T cell-APC contact area ( Figure 5C ). The colocalization of Pin1 and PKCθ was antigen-specific since it failed to occur when LG2 cells were fed with a mutated HA peptide possessing a single amino acid substitution (HA-K316E). The F-actin binding protein, phalloidin, served as an IS-specific marker in this study.