TCR crosslinking promotes the association of CypA with ZAP70
To identify CypA-binding partners in TCR-engaged T cells, which might play a role in T cell activation and are targets for regulation by CypA we immunoprecipitated CypA from lysates of resting and OKT3-stimulated Jurkat T cells and immunoblotted the samples with phospho-Tyr-specific mAbs. A prominent protein band of 70 kDa was observed in lysates of activated but not resting Jurkat T cells. Reblotting of the membrane with anti-ZAP70 mAbs indicated that this protein band corresponds to ZAP70 (Fig. 1A). To ascertain that the CypA-associated 70 kDa protein band represents ZAP70, we repeated the experiment using a ZAP70-deficient Jurkat T cell subline, P116. Anti-CypA mAbs were found to coimmunoprecipitate a ZAP70 immunoreactive protein from OKT3-stimulated Jurkat, but not P116 T cell lysates (Fig. S1A). In addition, heterologous expression of GST-CypA and Myc-ZAP70 in Jurkat T cells followed by anti-GST immunoprecipitation and anti-Myc immunoblotting reconfirmed the association between CypA and ZAP70 in OKT3-treated, but not resting T cells (Fig. 1B). An immunoprecipitation study in Jurkat T cells involving early and late time points of T cell activation demonstrated that binding of CypA to ZAP70 is transient and peaks at ~ 60 sec post-TCR crosslinking (Fig. 1C). Coimmunoprecipitation of ZAP70 with CypA was also observed in lysates of TCR-triggered C57BL/6J mouse spleen and thymus lymphocytes (Fig. 1D, E), suggesting that CypA binding to ZAP70 is a physiological phenomenon. Finally, a reverse coimmunoprecipitation study revealed the ability of ZAP70 to coimmunoprecipitate CypA from OKT3-stimulated Jurkat cells. (Fig. S1B).
To define the subcellular location of CypA-bound ZAP70 in TCR-engaged T cells, we stained the mouse spleen and thymus cells, as well as Jurkat T cells, with CypA- and ZAP70-specific Abs followed by a confocal microscope analysis. Cell staining with p-Tyr-specific mAbs verified the efficiency of the TCR stimulation by showing tyrosine-phosphorylated proteins at the membrane of the stimulated, but not resting Jurkat T cells (Fig. S1C). We found that in resting T cells, CypA and ZAP70 reside predominantly in the cytosol. In contrast, TCR crosslinking led to translocation and colocalization of the two proteins at the plasma membrane of the mouse spleen and thymus and Jurkat T cells (Fig. 1F-I, Fig. S1D-E).
Tcr Stimulation Promotes Cypa Binding To The Zap70 Interdomain B Region In An Lck-dependent Manner
TCR ligation triggers rapid phosphorylation of multiple effector proteins on tyrosine residues, hence we suspected that CypA-ZAP70 interaction might be regulated by one or more protein tyrosine kinases (PTKs). One of the major candidates for this phosphorylation is the Lck PTK which associates with the cytoplasmic tails of the CD4 and CD8 coreceptors. Lck recruits to the TCR/CD3, upon coreceptor binding to MHC, and phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) on the TCR/CD3 chains, as well as ZAP70 [40]. To test the involvement of Lck in CypA-ZAP70 interaction, we compared the ability of CypA to coimmunoprecipitate ZAP70 from wild-type Jurkat vs. the Lck-deficient Jurkat subline (JCaM.1). We observed that CypA coimmunoprecipitated ZAP70 from lysates of OKT3-treated Jurkat, but not JCaM.1 T cells (Fig. S2A). The results suggest that the in vivo interaction between CypA and ZAP70 is dependent on Lck, which is known to phosphorylate ZAP70 in TCR-triggered T cells [40]. Furthermore, we immunoprecipitated CypA from anti-CD3ε (2C11)-stimulated mouse spleen cells, and incubated the bead-immune-complexes in the presence or absence of calf-intestinal alkaline phosphatase (CIP) for 1 h. As observed above, CypA coimmunoprecipitated ZAP70 from lysates of 2C11-treated mouse spleen cells. However, CIP treatment of the immune complexes resulted in ZAP70 dissociation from the immune complexes (Fig. S2B) suggesting that CypA-ZAP70 interaction is dependent on the phosphorylation of ZAP70, CypA, or both proteins.
Previous studies demonstrated that CypA mediates its association with IL-2-inducible T-cell kinase (Itk), a predominant tyrosine kinase in T cells, by interacting with the N-SH2 domain in the Itk regulatory domain [41]. Hence, we speculated that CypA association with ZAP70 tyrosine kinase could also be through the ZAP70 regulatory region. A pull-down assay using various GST-ZAP70-deletion mutants demonstrated that CypA can be pulled down only by the full-length ZAP70 and the interdomain B region within the regulatory domain ZAP70 (Fig. 2A).
Association Of Cypa With Activated Zap70 Promotes Cypa Recruitment To The Tcr Within The Is
The ZAP70 PTK resides in the cytoplasm of resting T cells. Following TCR stimulation it translocates to the cell membrane and undergoes phosphorylation which peaks at ~ 60 sec [1]. Since CypA association with ZAP70 occurs in a similar time kinetic post TCR stimulation we tested whether the membrane-translocating ZAP70 pulls with it the CypA protein. Jurkat T cells were stimulated with OKT3 for 60 sec and their lysates were subjected to immunoprecipitation using phospho-CD3ζ (pCD3ζ)-specific Abs. As previously reported, ZAP70 and Lck coimmunoprecipitated with the pCD3ζ from TCR-activated T cells (Fig. 2B) [1, 42]. In addition, CypA was found to coimmunoprecipitate with pCD3ζ (Fig. 2B). CypA recruitment to the cell membrane was dependent on the presence of ZAP70 and did not occur in ZAP70-deficient P116 T cells (Fig. 2B). Immunofluorescence studies utilizing similar cells and activation conditions confirmed the colocalization of CypA and pCD3ζ at the plasma membrane of activated Jurkat T cells (Fig. 2C-F). To test whether the localization of CypA at the membrane of TCR-activated T cells occurs via its association with ZAP70 or perhaps by its direct interaction with pCD3ζ, the experiment was repeated using ZAP70-deficient P116 T cells. We found that the lack of ZAP70 disrupted the ability of CypA to coimmunoprecipitate with pCD3ζ (Fig. 2B) or colocalize with it at the plasma membrane (Fig. 2D), suggesting the requirement of ZAP70 for the association of CypA with the activated TCR.
We further speculated a tripartite complexing of CD3 receptor-ZAP70-CypA upon TCR crosslinking. In order to detect CD3 in both resting and activated cells, we utilized an antibody against total CD3 for the immunofluorescence studies. Since ZAP70 interacts with CD3ζ as well as CD3ε ITAM motifs [43] we used a combination of anti-CD3ε, -ZAP70, and -CypA Abs to immunostain OKT3-stimulated Jurkat T cells. While the three proteins were found to be distributed in the same subcellular compartments, stain overlapping analysis revealed a striking colocalization of CypA-ZAP70, ZAP70-CD3ε, and CypA-CD3ε -in OKT3-stimulated cells, in comparison to the staining results in resting Jurkat T cells (Fig. 3A-D). To test whether CypA recruits to the IS of T cells triggered by peptide antigen-loaded APC, by virtue of its interaction with ZAP70, we utilized CH7C17 Jurkat T cells, which express the influenza hemagglutinin (HA) peptide-specific TCR, and co-cultured them with antigen-fed LG2 cells, as APC. LG2 cells fed with the HA 307–319 peptide formed conjugates with the CH7C17 T cells in which CypA and ZAP70 colocalized at the T cell-APC contact area (Fig. 3E). The response was antigen-specific since colocalization of CypA-ZAP70 did not occur in LG2 cells fed with a mutated peptide, HA(K316E). CypA-ZAP70 also colocalized with the F-actin binding protein, phalloidin, which serves as an IS-specific marker. These results suggest a potential regulatory role for CypA in the vicinity of the ZAP70-TCR complex within the IS, where CypA might regulate IS-residing effector molecules that impact on TCR-downstream signaling cascades.
CypA inhibits ZAP70 catalytic activity in vitro
To test the direct effect of CypA on ZAP70 activity we performed an in vitro radioactive kinase assay on ZAP70 immunoprecipitates from 1 min OKT3-treated Jurkat T cells and 2C11-treated C57BL/6J mouse spleen and thymus T cells. Preincubation of ZAP70 from all three sources with enzymatically active recombinant CypA (rCypA) inhibited ZAP70 autophosphorylation activity in a concentration- (Fig. 4A, C, Fig. S3A, C) and time- (Fig. 4B, D, Fig. S3B, D) dependent manner. A linear CypA-mediated inhibition of the ZAP70 catalytic activity was observed in the three different sources of ZAP70 (Fig. 4E, F). Furthermore, an augmented ZAP70 kinase activity was noted in OKT3-stimulated CypA-deficient Jurkat T cells when compared to that of wild-type cells (Fig. 4G, H). These data suggest that CypA functions as a negative regulator of ZAP70 enzymatic activity.
To test whether ZAP70 is sensitive to isomerases in general, or whether its regulation is selective to CypA, we repeated the assay comparing the effects of CypA vs. Pin1 PPIase on the catalytic activity of ZAP70. We found that CypA, but not Pin1, downregulated the autophosphorylation activity of ZAP70 (compare Fig. S4A vs. S4B). A control experiment validated that rPin1 is catalytically active by showing its ability to modulate the activity of PKCα, a known Pin1 substrate [44] (Fig. S4C). Inclusion of a ZAP70 substrate, the cytoplasmic fragment of human erythrocyte band 3 (cfb3) [45] in the in vitro kinase assay further demonstrated that rCypA inhibited the ability of ZAP70 to phosphorylate an exogenous substrate in a concentration- (Fig. S3C) and time- (Fig. S3D) dependent manner. We also noticed that the inclusion of recombinant CypA in the ZAP70 kinase assay system did not result in phosphorylation of CypA, negating the possibility of reciprocal regulation of CypA by ZAP70 (Fig. 4, Fig. S3). While the above studies suggest that CypA inhibits the phosphorylation activity of ZAP70, they do not rule out the possibility that the reduction in ZAP70 phosphorylation is due to rCypA-mediated dephosphorylation of ZAP70. This hypothesis was tested by co-incubation of enzymatically active rCypA with an inactive, radiolabeled, phospholabeled ZAP70. We found (Fig. S4D) that rCypA was unable to dephosphorylate ZAP70, supporting the assumption that CypA is a conformational regulator of ZAP70.
Csa Reverses The Regulatory Effect Of Cypa On Zap70
CsA is a potent immunosuppressive drug that mediates high-affinity binding to CypA and inhibits its enzymatic activity [46, 47]. To test whether CypA binding to ZAP70 is affected by CsA, we treated C57BL/6J-derived spleen and thymus cells and Jurkat T cells with CsA and subsequently stimulated the cells with anti-CD3 (2C11/OKT3) mAbs, followed by immunoprecipitation of CypA. We found that cell treatment with CsA inhibited the ability of CypA to associate with ZAP70 (Fig. 5A, Fig. S5A, S6A). The effect of CsA on the ability of CypA to associate with ZAP70 was also tested by cell staining and immunofluorescent imaging using confocal microscopy. The results demonstrated strong CsA-mediated inhibition of CypA colocalization with ZAP70 in C57BL/6J mouse spleen and thymus cells and Jurkat T cells (Fig. 5B-D, Fig. S5B-D, S6B-D). A major mechanism by which CsA-CypA complexes contribute to immunosuppression is by binding to and inhibition of calcineurin and its downstream signaling pathway that controls IL-2 gene transcription. However, the above findings also suggest the involvement of CypA in the regulation of ZAP70. We tested the effect of CsA on the ability of rCypA to attenuate ZAP70 catalytic activity. The inclusion of CsA at the rCypA-ZAP70 preincubation step reversed the inhibitory effect of CypA on ZAP70 autophosphorylation in a concentration-dependent manner in C57BL/6J mouse spleen and thymus cells and in Jurkat T cells (Fig. 5E, Fig. S5E, S6E). The results suggest that CsA-mediated regulation of CypA in activated T cells may affect the catalytic activity of ZAP70 and potentially modulate signal transduction pathways downstream of the TCR.
Previous studies demonstrated that CypA is a negative regulator of the Itk in T cells and that CsA can augment the tyrosine phosphorylation of PLCγ1, the primary substrate of Itk [41]. In analogous to this system, we tested the effect of CsA on ZAP70-mediated phosphorylation of LAT, the immediate physiological substrate of ZAP70 in TCR-stimulated T cells [15]. Immunoprecipitation of LAT from OKT3-treated Jurkat T cells revealed its rapid phosphorylation at 60-sec post-TCR/CD3 stimulation. Pretreatment of the cells with CsA augmented the tyrosine phosphorylation of LAT (Fig. 5F). CsA had no effect on the phosphorylation level of LAT at a longer temporal post-TCR stimulation (10 min) when the amount of ZAP70-associated CypA was negligible. Higher tyrosine phosphorylation of LAT was also observed in TCR/CD3-stimulated CypA-deficient Jurkat T cells, relative to the phosphorylation level observed in wild-type cells (Fig. 5G). These results suggest that ZAP70-mediated phosphorylation of LAT in vivo, which occurs at an early time-point post TCR stimulation, is subjected to regulation by CypA.
To substantiate the latter findings showing in vivo effects of CypA/CsA on ZAP70-mediated LAT phosphorylation, we transfected Jurkat T cells with the ROZA-XL plasmid which functions as a biosensor of ZAP70 activity (see Fig. S7). FACS analysis of ROZA-XL-expressing cells revealed that OKT3 stimulation led to a decrease in their FRET values which reflects increased ZAP70 activity (Fig. 5H). No change in FRET was observed in OKT3-stimulated cells that express the ROZA-XL-YF plasmid which encodes a ZAP70 activation-insensitive protein [48]. To ensure the accuracy of FRET readings, the transfection efficiency of ROZA-XL/YF was validated by FACS analysis, and equal numbers of ROZA-XL/YF positive cells were used in each experimental group. Interestingly, pretreatment of the ROZA-XL-expressing cells with CsA further reduced the OKT3 stimulation-induced FRET values, suggesting that inhibition of CypA at an early time-point post TCR stimulation increases the activity of ZAP70 (Fig. 5H). The results suggest the involvement of CypA in the regulation of an early T cell activation response which leads to downregulation of ZAP70 catalytic activity, an effect that can be abrogated by the CypA inhibitor, CsA.