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

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.


Introduction
Protein kinase C q (PKCq) is highly expressed in T lymphocytes and is sine qua non for T cell antigen receptor (TCR)-coupled signal transduction which eventually leads to cell activation and proliferation. Importantly, PKCq is non-redundant with other T cell-expressed PKC isoforms and is unique in its ability to translocate to a defined central sub-domain of the immunological synapse (IS) in antigen-stimulated T cells (1)(2)(3)(4)(5)(6). Signals delivered by the TCR and the CD28 costimulatory receptor induce membrane translocation and kinase activation of PKCq (7). As a critical component of the TCR/CD28-coupled signaling machinery, PKCq has the ability to integrate multiple signals and fine-tune signaling pathways required for a specific output. It is subjected to regulation by multiple mechanisms and undergoes phosphorylation at several sites that determine its spatiotemporal conformation and catalytic activity, as well as its ability to interact with cofactors, substrates, and other binding proteins. Although the TCR stimulation-induced PKCq recruitment to the IS is well established (4, 8), the underlying molecular basis has remained elusive for a long time. Hitherto, we provided a mechanistic explanation for this selective process by showing that the PKCq-V3 polyproline-rich region, a phylogenetically conserved sequence across species (9), is involved in the localization of PKCq to the center of the IS, which is mediated by indirect association with the cytoplasmic tail of the costimulatory receptor CD28 (9). Despite the importance of PKCq in T cell activation, the entire spectrum of mechanisms that regulate the kinase is still lacking.
Proline-directed protein phosphorylation has a crucial role in diverse cellular processes and signal transduction pathways. It is one of the most frequent post-translational modification sites in proteins (10). Proline exists in one of two distinct forms that acquire either cis or trans conformations. The cis-trans interconversion of Pro in peptides can occur spontaneously in an aqueous solution at a very slow rate. However, peptidylprolyl isomerases (PPIases) can rapidly interconvert the cis-trans isomers of peptide bonds with the amino acid proline. PPIases can modulate protein topology by catalyzing a cis-trans switch at appropriate Xxx-Pro motifs (where Xxx indicates any amino acid), thereby regulating the reversible transition of substrate proteins between active and nonactive isomers (11). The PPIases are grouped into three families namely: cyclophilins, FK506-binding proteins (FKBPs), and parvulins. These enzymes assist in the folding of newly synthesized proteins and regulate the stability, localization, and activity of mature proteins. For instance, works from our lab have established PPIases regulation on signaling proteins CT-10 regulator of kinase (Crk) and zeta chain associated protein (ZAP-70) (12)(13)(14). They also regulate gene transcription at multiple levels. The phospho-Ser/Thr-Pro (pS/T-P) motifs in proteins are thermodynamically hindered and render inaccessible to conventional PPIases, such as cyclophilins and FKBPs. In contrast, the pS/T-P bond serves as a specific recognition motif for Pin1, a member of the parvulin family, which can cis/trans isomerize the proteins and regulate their stability and activity (15). Pin1 regulates the activity of various transcription factors that are essential for T cell activation, including NF-kB and AP1. It also regulates the activity of protein kinases and phosphatases at the plasma membrane, cytosol, and nucleus (16,17). A report by Abrahamsen et al. suggests that Pin1 provides a 'timer' for the lifetime of conventional PKC (cPKC) isoforms by converting them into a species that can be readily dephosphorylated, ubiquitinated, and degraded following cell activation by agonists (18). In these studies, the regulation by Pin1 required both the catalytic domain of the isomerase and the presence of a Pro immediately following the phosphorylated Thr at the turn-motif phosphorylation site, one of two C-terminal sites that are phosphorylated during the maturation of PKC isozymes (18).
Human PKCq possesses seven Ser/Thr-Pro motifs that theoretically, upon phosphorylation, could serve as putative Pin1binding or -regulatory sites. In the present work, we demonstrate the significance of the phospho-Thr 335 -Pro motif within the V3 polyproline-rich region of the PKCq regulatory domain, which functions as a critical priming site for the activation of PKCq, its binding to CD28 and subsequent IS localization. We highlight the potential role of the TCR stimulation-induced transient phosphorylation of the Thr 335 -Pro motif in the spatiotemporal regulation of PKCq and in the temporal association between PKCq and the PPIase Pin1. TCR/CD3 crosslinking or co-culture of antigen-specific T cells with antigen-pulsed APCs led to the colocalization of PKCq and Pin1 at the T cell membrane or at the IS, respectively. Together, we portray Pin1 as a novel binding partner of PKCq in activated T cells.

Animals, cell lines, culture conditions, and cell stimulation
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.
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.
PKCq mutants [T/A, A 335 P; T/E, E 335 P] were generated by sitedirected mutagenesis and overlapping PCR.

Nucleofection of Jurkat T cells, and transient transfection of eukaryotic cells
Non-adherent cells: Jurkat T cells maintained at a logarithmic phase were washed in supplement-free RPMI 1640, resuspended either in 700 ml transfection buffer (1.8 g/ml sucrose, 2nM DTT in RPMI 1640) or in 250 ml Mirus Ingenio ® electroporation solution and transferred into 0.4 cm-gap Gene Pulser cuvettes (BioRad) (20x10 6 cell/700 ml/cuvette). Plasmid DNA (10 mg/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% FCScontaining DMEM (2x10 6 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 (5mg/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 cm 2 tissue culture flasks for 48h.
Cell lysate preparation, immunoprecipitation, and immunoblotting 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 Na 3 VO 4 , 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.
GST fusion proteins and pull-down assay pGEX bacterial expression vectors were used to transform Escherichia Coli DH5a ™ 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 mg/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.

Preparation of mouse thymus tissue sections
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.

Immunofluorescence staining and confocal microscopy
Jurkat T cells (2×10 6 /group) were seeded on poly-L-lysinecoated 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-PKCq 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 546conjugated anti-mouse (or rabbit) Ig and Alexa Fluor 488conjugated 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-PKCq 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 nonspecific 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 PKCq was quantified using the JACoP ImageJ plugin (21).

Conjugate formation assay and immunofluorescence
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 PKCq, 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 mg/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-PKCq and rabbit anti-Pin1, followed by incubation with Alexa Fluor 633-conjugated phalloidin and: Alexa Fluor 488conjugated anti-rabbit IgG and Alexa Fluor 546-conjugated antimouse IgG. CytoPainter was detected at 405nm (Olympus FluoView FV1000 laser-scanning confocal microscope).
In silico modeling of the proposed PKCq-Pin1 complex Using the Swiss-PdbViewer package (23), we modeled the complex of the WW domain of Pin1 with PKCq 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-pSer 174 -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-Thr 335 -Pro-Gly peptide of PKCq on the bound peptide, with pSer 174 -Pro and pThr 335 -Pro respectively as anchors. The major binding pocket in the crystal structure includes Ser 16 , Arg 17 , and Ser 18 that coordinate the phosphate group and Trp 34 and Tyr 23 that line a hydrophobic pocket that fit the proline side chain. The corresponding PKCq moieties fit this pocket in the model by construction, as they adopt the coordinates of almost identical residues. Tyr 23 also lines a minor binding pocket, together with Phe 25 . 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 PKCq residues in the model does not confer any clashes or incompatibility.

Retroviral shRNA silencing of Pin1 in Jurkat T cells
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 mg) 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 (2x10 5 ) 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
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.

Results
Phosphorylation of Thr 335 -Pro is critical for PKCq activation, binding to partners, and IS localization PKCq 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 PKCq regulatory domain was recently found to be necessary and sufficient for PKCq IS localization and function (9). Moreover, human PKCq harbors several Ser/Thr-Pro motifs that are predicted to be phosphorable, posing them as additional potential regulatory sites. Among them, Thr 335 -Pro was of particular interest, owing to its location within the unique PR hinge region of PKCq ( Figures 1A-C). To test this conjecture, mutagenesis of this key Thr 335 residue, followed by PKCq-related functional aspects has been employed. Luciferase reporter assay and coimmunoprecipitation studies were utilized to test whether Thr 335 plays a role in PKCq 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 Thr 335 point mutants [T/A, A 335 P; T/E, E 335 P]. For the reporter assay, the cells were cotransfected with a luciferase reporter for PKCqdownstream effector molecules, including NFAT and NF-kB, or the CD28 response element RE/AP, and tested following TCR crosslinking using anti-CD3/CD28 mAbs. For the binding assay, PKCq wild-type or the Thr 335 mutant-overexpressing Jurkat T cells were lysed and subjected for coimmunoprecipitation using anti-CD28 mAbs. Our results demonstrated that phosphorylation of Thr 335 is required for the activation of PKCq (Figures 1D-G) and its binding to the costimulatory receptor, CD28 ( Figure 1H). Thr 335to-Ala amino acid substitution completely abolished the ability of PKCq to be triggered or to bind CD28, while replacement of Thr 335 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-Thr 335 is required for PKCq localization at the IS ( Figures 1I, J). These data highlight the potential role of the Thr 335 -Pro motif in the spatiotemporal regulation of PKCq and suggest that regulation via this motif is phosphorylation-dependent.
Pin1 mediates a phosphorylationdependent binding of PKCq, and the integrity of the PKCq-V3 is a requisite for this interaction Based on our preliminary observations, we hypothesized that the phosphorylation of Thr 335 -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 PKCq that can bind Pin1, we used a heterologous cell system involving COS-7 cells transfected with different Xpress-tagged PKCq 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 PKCq in its ability to interact with the Pin1 protein ( Figure 2B).
PKCq is subjected to post-translational modifications, primarily by phosphorylation of multiple sites (25). To study whether Pin1 interaction with PKCq 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 PKCq that coimmunoprecipitated with anti-Pin1 mAb almost completely dissociated from PKCq following the treatment with calf intestinal phosphatase (CIP) ( Figure 2C) or l protein phosphatase (lPP) ( Figure S1A). The results indicate that interaction between the two proteins is phosphorylationdependent. Finally, by utilizing an appropriate isotype control, we validated the specificity of PKCq coimmunoprecipitation with Pin1 ( Figure S1B).

Pin1-PKCq interaction is mediated by the Pin1 N-terminal WW domain and the PKCq pThr 335 -Pro motif
Pin1 has a modular domain architecture comprising an Nterminal 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, R 17 A, in the WW domain of Pin1 abolishes the ability of Pin1 to interact with its known substrates, whereas a substitution mutation, C 113 A, downregulates the Pin1 catalytic activity. To determine the Pin1 domain that interacts with PKCq we used bead-immobilized GST fusion proteins that include Pin1 without or with substitution replacement in the WW (R 17 A) or catalytic (C 113 A) 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 PKCq ( Figure 3C). The data indicate that the integrity of the WW domain, but not the PPIase domain, is essential for Pin1-PKCq interaction in T cells.
Since we showed that PKCq-V3 is required for Pin1 binding (Figure 1), we extended our studies on whether the Thr 335 -Pro motif within PKCq-V3 could be integral for the binding of Pin1. Hence, we conducted a study in COS-7 cells that were transfected with PKCq WT, and mutants T 335 A, and T 335 E, immunoprecipitated the transiently expressed PKCq from their lysates (using anti-Xpress mAbs) and tested for Pin1 coimmunoprecipitation. As expected, Pin1 coimmunoprecipitated with PKCq from the lysate of COS-7 cells transfected with PKCq WT. However, replacement of Thr 335 by Ala completely abolished the ability of PKCq to bind Pin1 whereas substitution of Thr 335 by the Glu phosphomimetic, reinstated Pin1 binding ( Figure 3D). The data shows that phosphorylation of the Thr 335 -Pro motif in the PKCq-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 PKCq-V3-derived amino acid sequence that includes Thr 335 plus 10 adjacent amino acids on each side. While both peptides had identical sequences, only pKS21-Myc was phosphorylated on Thr 335 . 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 PKCq-V3 on the Thr 335 -Pro motif to turn PKCq into a Pin1 binding partner ( Figure 3E). In addition, in silico structural modeling analysis revealed a suggestive interaction between PKCq-Thr 335 -Pro and the critical amino acid residues in the Pin1-WW binding domain (Arg 17 , Tyr 23 , and Trp 34 ) to materialize a PKCq-Pin1 complex ( Figure 3F and Figure SV1). Overall, these results demonstrate that Pin1-PKCq interaction is mediated by the Nterminal WW domain of Pin1 and the PKCq-pThr 335 -Pro motif.

T cell activation-induced binding of Pin1 to PKCq in C57BL/6J-derived splenocytes and Jurkat T cells follows a temporal time kinetic, whereas interaction is constitutive in mouse thymocytes
To further analyze the ability of Pin1 to interact with PKCq 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 PKCq coimmunoprecipitation. We found that PKCq 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, PKCq interacted with Pin1 at basal level at all time points post-TCR-crosslinking ( Figure 4C). The results show that association of Pin1 with PKCq in TCR-triggered splenic T cells and Jurkat T cells occurs in a time kinetic which corresponds to that of the recruitment of PKCq to the IS. The constitutive binding of Pin1 to PKCq 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-PKCq interaction was further validated by demonstrating the ability of GST-Pin1 to pull down PKCq from TCRstimulated Jurkat T cells ( Figure 4D). We also analyzed the selectivity of the Pin1 PPIase to PKCq by comparing the ability of other PPIases (CypA and FKBP12) to associate with it. Indeed, we found that Jurkat T cell-derived PKCq selectively associates with Pin1 but not with other peptidyl-prolyl cis-trans isomerases ( Figure S2B). Finally, we found that Pin1-PKCq association can also be demonstrated using a reciprocal immunoprecipitation, where anti-PKCq mAbs coimmunoprecipitate Pin1 from lysates of OKT3-stimulated Jurkat cells ( Figure 4E).

TCR engagement promotes PKCq and Pin1
clustering at the plasma membrane within the immunological synapse PKCq 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 PKCq-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 fluorescentlylabeled Pin1-and PKCq-specific Abs. Cell analysis using confocal microscopy demonstrated an augmented association between Pin1 and PKCq at the cell membrane ( Figure 5A, and Figure S3A). Previous observations showing a constitutive association between Pin1 and PKCq 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 PKCq 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 PKCq ( Figure S3B). The results corroborate the interaction of Pin1 and PKCq at the membrane of T cells and hint for a possible involvement of Pin1 in the regulation of PKCq and PKCq-dependent early activation events in TCR-engaged T cells.
Since engagement of the TCR promotes PKCq translocation to the center of the IS, we further analyzed whether the same applies for the PKCq-bound Pin1 protein. We used Jurkat T cells (clone CH7C17), which express the influenza hemagglutinin (HA) peptide-specific Vb3 TCR, and co-cultured them with antigen-fed LG2 B cells, as APC. Cell staining with fluorescently-labeled Pin1-and PKCq-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 PKCq colocalized at the T cell-APC contact area ( Figure 5C). The colocalization of Pin1 and PKCq 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.

Discussion
In the past three decades, the role of PKCq in T cells was intensively studied. It was initiated in 1993, when the human PKCq gene was originally cloned (29), yielding many important findings along these years (30)(31)(32). The present work focuses on the Thr 335 -Pro motif within the PKCq-V3 regulatory domain and its identification as a key priming site for the IS localization and activation of PKCq. Moreover, we demonstrated that Thr 335 -Pro could serve as a docking site for the Pin1 enzyme, which specifically recognizes phospho-Ser/Thr-Pro motifs (10,33).
PKCq is subjected to allosteric regulation and post-translational modifications, primarily by phosphorylation at multiple sites (25). For example, phosphorylation of Thr 538 at the activation loop (34), is mediated by the GLK kinase (35) and is critical for PKCq activation and TCR downstream signaling (25). The role of other potential phosphorylation sites is not fully understood. Recently, a phospho-Ser 241 -Pro-Thr motif in PKCq was predicted to function as a nuclear localization signal (NLS) (36), while our present results suggest that Thr phosphorylation within the Thr 335 -Pro-Gly motif is required for the IS localization and activation of PKCq. The selective localization of the PKCq isoform is mediated by a direct or indirect association between its V3 domain and the cytoplasmic tail of CD28, the prototypic costimulatory receptor on T cells. This interaction is mediated by the Lck protein tyrosine kinase (PTK) that associates, via its SH2 and SH3 domains, with a tyrosinephosphorylated CD28 motif and a PR sequence in the PKCq V3 region, respectively (9). Here, we elucidate, for the first time, the mechanistic basis for the selective, non-redundant IS localization and function of PKCq in T cells.
Herein, we demonstrate that Thr 335 -Pro in the PR motif within the PKCq-V3 domain is a TCR-stimulation-dependent phosphorylation site which is critical for PKCq recruitment and localization at the IS, a prerequisite for the induction of TCR-linked signal transduction pathways. Substitution of Thr 335 by Ala completely abolished PKCq activation and its ability to bind CD28, while Thr 335 replacement by Glu, which mimics the approximate size and charge of phospho-Thr, restored these properties. Fluorescent cell staining and confocal microscopy confirmed that phospho-Thr 335 -Pro is required for IS localization of PKCq. Our studies highlight the potential role of the Thr 335 -Pro motif in the spatiotemporal regulation of PKCq and corroborate the assumption that regulation via this motif is phosphorylation-dependent.
Furthermore, we highlight phospho-Thr 335 -Pro as a putative site for association with the Pin1 PPIase. Pin1 association with PKCq in TCR-triggered T cells is dependent on the phosphorylation state of PKCq. Interaction between these two proteins is hampered by the presence of active phosphatases (CIP and l PP), supporting the assumption that phosphorylated Ser/Thr residues in PKCq are required for the interaction with Pin1.
Pin1 possesses two major functional domains; an N-terminal substrate-binding WW domain (amino acids 6-37) and a C-terminal PPIase catalytic domain (amino acids 54-163). We found that a single amino acid substitution mutation, R 17 A, in the WW domain of Pin1 abolished the ability of Pin1 to interact with PKCq. In contrast, a substitution mutation, C 113 A, which downregulates the Pin1 catalytic activity had no effect on its ability to interact with PKCq. Therefore, it appears that the integrity of the WW domain, and not the catalytic domain of Pin1 is essential for interaction with PKCq in activated T cells.
Based on previous studies, showing that the PKCq Thr 335 -Pro-Gly motif is required for the activation and association of PKCq with CD28, we hypothesized that the phospho-Thr 335 -Pro-Gly sequence might serve as a binding site for Pin1. Utilizing a heterologous system of COS-7 cells that were transfected with wild type PKCq, or PKCq mutants we found that the Thr 335 -Pro-Gly motif of PKCq is essential for Pin1 binding. The utilization of PKCq-deficient COS-7 cells eliminated the influence of endogenous PKCq in the mutant protein coimmunoprecipitation studies. A plausible structural interpretation of this observation is supported by a computational model of the PKCq-V3-derived short peptide, in association with the Pin1-WW domain.
Previous studies demonstrated that Pin1 can interact with and downregulate the activity of cPKC isoforms (18). In these studies, COS-7 cells were cotransfected with the relevant PKC isoform plus GST-Pin1, and PKC-Pin1 binding was evaluated by GST pull down and immunoblot. In contrast to the cPKC isoforms that were easily detected in the pull-down assay, only a small fraction of the cellular PKCq was pulled down by GST-Pin1. In the present study, we pulled down PKCq from Jurkat T cell lysates using bead-immobilized GST-Pin1 and Pin1 mutants and observed a relatively strong association between the two proteins. The in vivo association between PKCq and Pin1 was further substantiated using mouse splenic T cells as well as by coimmunoprecipitation and cell staining analyses. Although the Pin1 isomerization site in the cPKC isoforms is missing in PKCq (18), we found that PKCq possesses a distinct Pin1 binding site within its regulatory regions, which is absent in cPKC isoforms.
The current binding studies performed in T cells from different sources, including human Jurkat T cells and freshly isolated mouse splenic T cells, revealed a transient association between PKCq and Pin1 that was augmented following TCR crosslinking. In contrast, a cell activation-independent constitutive association between PKCq and Pin1 was observed in thymic T cells, which include a high proportion of proliferating and differentiating cells that undergo positive and negative selection in response to triggers by thymic epithelial cells and the thymic microenvironment. A constitutive interaction between Pin1 and PKCq was also observed in fluorescently labeled thymocytes in thymic sections derived from C57BL/6J mice. The thymus consists of heterogeneous cell populations that upregulate distinct kinase activities during different stages of differentiation and maturation (37)(38)(39), and at a certain stage (CD4 + CD8 + double positive cells) also form multifocal immunological synapse with sustain tyrosine phosphorylation (40). While functionally active PKCq is known to be essential for the positive selection of thymocytes (41), expression of functinal Pin1 in thymocytes appears to be as important, since its ablation disrupts their genetically predetermined mechanisms of differentiation and maturation (42).
The rapid and transient interaction between PKCq and Pin1 follows a time kinetic that corresponds to that of PKCq recruitment to the IS, suggesting that Pin1 might play a role in the regulation of PKCq recruitment to the IS, and in activation of the PKCqdependent early activation events in TCR-triggered T cells. Members of each of the three families of PPIase (cyclophilins, FKBPs, and parvulins) regulate the conformation, stability, localization, and activity of distinct protein kinases (43-46). We demonstrated that PKCq interacts with Pin1, but not other PPIases (CypA or FKBP12) in TCR-stimulated T cells, demonstrating the selectivity and specificity of Pin1 to PKCq.
In resting T cells, PKCq resides predominantly in the cytosol while majority of the Pin1 proteins are confined to the nucleus (2,27,28). Immunofluorescence imaging of TCR/CD3-activated Jurkat T cells showed enhanced recruitment of Pin1 and PKCq to the plasma membrane, where the two proteins colocalized, providing further support for the in vitro results showing reciprocal coimmunoprecipitation of the two proteins. Strong colocalization of Pin1-PKCq was visualized at the heterotypic cell contact area, and since PKCq is known to localize to the center of the IS, the results suggest that Pin1 in TCR-activated T cells is an integral constituent of the IS. To further support the hypothesis that Pin1 colocalize with PKCq at the IS we utilized antigen-specific T cells and induced their activation using antigen-fed APC. This system included Jurkat T cells, clone CH7C17, which express a Vb3 TCR specific for the influenza HA 307-319 peptide, and LG2 B cells, as APC, that were fed with the specific HA peptide or with a mutated peptide that possess a single amino acid substitution (HA-K316E). Coincubation of the two cell types followed by their staining with fluorescently-labeled Pin1-and PKCq-specific Abs and analysis using a confocal microscope revealed that in a fraction of cells that formed conjugates, Pin1 and PKCq colocalized at the IS, at the T cell-APC contact area, that was co-stained by the fluorescentlylabelled F-actin binding protein, phalloidin, which serve as an ISspecific marker. The results highlight the possibility that Pin1 functions as a molecular timer that finetunes PKCq phosphorylation and activation and contributes to the regulation of its downstream effector molecules.
Previous studies demonstrated that PKCq IS localization requires the interaction of PKCq with the cytoplasmic tail of CD28 (9,31). This indirect interaction is made possible by Lck which utilizes its SH2 and SH3 domains for the simultaneous interaction with a PYAP motif in CD28 and a PxxP motif in PKCq, respectively. The sites of interaction of Lck (P 331 CLP) and Pin1 (T 335 PG) in PKCq are extremely close, suggesting a potential binding competition between Pin1 and Lck. However, Pin1 and Lck might also interact with PKCq independent of each other, or in a sequential manner. For example, Pin1 interaction with PKCq might define the conformation of the latter, which will determine the accessibility of the PKCq P 331 xxP motif for interaction with Lck, and consequently, the ability of the Lck-PKCq complex to recruit to the IS. Furthermore, the competitive, independent, or sequential binding of Pin1 and Lck to PKCq may differ among T cell subtypes and thereby explain the differential recruitment of PKCq to the IS of Th/Tc vs its recruitment to the opposite cell pole in Treg.
Preliminary studies in Pin1 shRNA-expressing Jurkat T cells revealed that knock-down of Pin1 downregulates NF-kB reporter activity in TCR stimulated cells ( Figure S4). However, since Pin1 regulates the conformation and activity of multiple proteins, these findings could reflect either a direct effect of Pin1 on PKCq or on PKCq downstream effectors that regulate the activity of NF-kB.
The current study identified a previously undescribed signature motif in the PKCq modular structure, which is required for PKCq IS localization and activation. Furthermore, TCR-stimulationinduced phosphorylation of this motif dictates its ability to interact with Pin1. Together, we report on a novel interaction between two enzymes-PKCq and Pin1, which might contribute to the amplitude, duration, and fine-tuning of the T cell activation response.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement
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.

Author contributions
NI conceived the study, designed experiments, and supervised the project. NPA, AM, PRN, and K-FK carried out the experiments. CK conducted the structural studies. ZS, AB, AA, and NI analyzed the data. EL and AA provided essential tools for the study. NPA, and NI wrote the manuscript. All authors commented on the previous versions of the manuscript. All authors contributed to the article and approved the submitted version.