In this study, we demonstrate that inducible CHC phosphorylation occurs as a result of TCR signaling after TCR engagement in both CD4+ and CD8+ T cells. The Src family kinase Lck is apparently responsible for CHC phosphorylation and its activity is influenced by the integrity of the downstream signaling molecule ZAP-70. CHC phosphorylation positively correlates with ligand-induced TCR internalization in both CD4+ and CD8+ T cells and with constitutive TCR endocytosis in CD4+ T cells. Pervanadate treatment causing irreversible CHC phosphorylation reduced both constitutive and ligand-induced TCR internalization in CD4+ T cells, and this inhibition occurred at the early stages of ligand-induced TCR endocytosis from the plasma membrane. By correlation, these studies implicate both CHC phosphorylation and dephosphorylation as influential in TCR internalization pathways.
Receptor-mediated endocytosis can be regulated both by modification of the cytoplasmic domains of internalized receptors and by receptor signaling effects on clathrin coat components. For a number of receptors including EGFR and CTLA-4, ligand binding causes tyrosine kinase activation, resulting in a structural change that exposes adaptor-binding motifs in the cytoplasmic domain of the receptors (18
). Furthermore, receptor signaling induces the modification of trafficking proteins, in particular the phosphorylation of the CHC (20
). It has been proposed that upon TCR engagement, the phosphorylation of the ITAMs within the cytoplasmic domains of the TCR–CD3 complex induces a conformational change exposing adaptor-binding motifs allowing for the assembly of the CCV and TCR internalization (10
). Here we demonstrate that TCR signaling also induces CHC phosphorylation in activated CD4+
T cells. Similar to the EGFR and the BCR (20
), CHC phosphorylation upon TCR engagement is mediated by a Src family protein tyrosine kinase. Our results indicate that the Src family kinase Lck directly phosphorylates CHC in vitro, and in Jurkat cells deficient in Lck, inducible CHC phosphorylation is abolished. Although Lck plays a critical role in CHC phosphorylation, we demonstrate that its activity is influenced by the integrity of the proximal TCR signaling cascade. The reduction in CHC phosphorylation observed in Jurkat T cells defective in the downstream kinase ZAP-70 suggests that the activation of this kinase positively regulates Lck activity, thus explaining its influence on CHC phosphorylation and correlation of ZAP-70 activity with TCR internalization.
In this study, we demonstrate a correlation between CHC phosphorylation by Lck and TCR internalization after activation of both CD4+
T cells by receptor engagement. Our data also suggest that basal Lck activity plays a role in constitutive TCR internalization. Interestingly, the CD8+
CTL clone we examined here exhibited very minimal constitutive TCR internalization, and its ligand-induced TCR endocytosis was significantly less than the CD4+
Jurkat cells despite having higher overall levels of the receptor on its surface. Recently, constitutive versus ligand-induced TCR down-modulation has been evaluated in murine CD4+
T cells (10
), but our study is the first to examine these processes in a human CD8+
CTL clone. Although our findings may simply reflect intrinsic differences in normal versus transformed cells, we speculate that the discrepancies in constitutive versus ligand-induced TCR internalization in the CD4+
T cells might be attributed to the different specialized functions of these cells in vivo.
Previously, the site of clathrin phosphorylation upon EGFR signaling was mapped to a single tyrosine residue at amino acid position 1477 (20
) in the region of the heavy chain that interacts with light chain and regulates assembly of the clathrin triskelion (38
). Our preliminary studies suggest that this tyrosine is also a target for Lck phosphorylation of CHC as well as adjacent tyrosine residues (unpublished data). It has been proposed that CHC phosphorylation could act either as a positive or a negative regulatory signal for clathrin function. For example, BCR signaling activates the Src family kinase Lyn necessary for CHC phosphorylation. This phosphorylation was shown to correlate with BCR internalization after signaling in rafts. The effect of CHC phosphorylation was compatible with positive stimulation of assembly of clathrin associated with rafts. It was, however, also compatible with a “negative” stabilization of preformed coated pits. In this latter scenario, phosphorylation might delay interactions with regulatory proteins required for the pit to vesicle transition to capture spent signaling receptors from rafts (21
). In this study, we report the first data suggesting that CHC phosphorylation may indeed serve as a negative regulatory signal for CCV formation. Although our data show that CHC phosphorylation correlates with TCR internalization, we also demonstrate in experiments with pervanadate that if CHC dephosphorylation cannot occur, both constitutive and ligand-induced TCR internalization are reduced. This inhibition of TCR internalization occurs at the plasma membrane, reducing access of the TCR to early endosomes. Although pervanadate has pleiotropic effects on cells, we found that CHC phosphorylation in the Lck-deficient Jurkat cells treated with pervanadate was significantly reduced compared with wild-type Jurkat cells, suggesting that CHC phosphorylation after either pervanadate treatment or TCR engagement involves Lck activity. Furthermore, we demonstrated that the internalization of the transferrin receptor was not inhibited by pervanadate treatment, thus eliminating the possibility of pleiotropic effects of this drug on factors that affect endocytosis in general. Thus, we propose that CHC phosphorylation and dephosphorylation are relevant to signaling receptors whose internalization is induced by ligand but not those that are constitutively internalized.
Similar to its proposed role in BCR endocytosis, CHC phosphorylation could influence clathrin function during the initial TCR internalization from the plasma membrane once the receptors have completed their signaling in rafts. However, considering what is known about TCR pathways of down-modulation, it is also possible that CHC phosphorylation plays a role in the regulation of receptor levels at an additional intracellular site. Before encounter with antigen, TCRs are continuously internalized and recycled back to the cell surface (2
). After TCR engagement with antigen, the TCR is internalized and trafficked to the lysosome for degradation (8
). Clathrin is involved in targeting receptors destined for degradation from the endosome to lysosomes, and this pathway involves both ubiquitination of receptors and the adaptor molecule Hrs that interacts with ubiquitin and clathrin (15
). CHC phosphorylation could be active during both internalization from the plasma membrane and in diversion from endosomes to lysosomes. Further analysis will be required to establish how this modification of CHC influences CHC-interacting proteins and affects either or both of these two clathrin-mediated pathways.
Recent studies have established the importance of TCR signaling (for review see reference 49
) and TCR down-modulation (50
) at the contact site between T cells and APCs, termed the immunological synapse. TCR down-modulation at this synapse serves two purposes. Internalization of the TCR from the plasma membrane to the endosome potentiates T cell activation by bringing the receptor in close proximity to critical molecules required for progression of the TCR signaling cascade (7
). The ultimate result of these signaling events is the complete activation of the T cell and the development of an effective T cell response to pathogenic organisms, tumors, or self-ligands in cases of selection and autoimmunity. Second, TCR down-modulation attenuates signaling by targeting the TCR from the endosome to a lysosomal compartment for degradation (8
). This process maintains a homeostatic balance between activation and down-modulation of the T cell response and may also function as a mechanism by which tumors induce lack of detection by the immune system (51
). Clathrin has been implicated in both of these regulatory steps, and here we demonstrate that TCR signaling can influence clathrin modification after T cell activation through phosphorylation and dephosphorylation. We propose that this modification plays a role in both the development and the modulation of the T cell response.