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. 2003 Mar;23(6):2017-28.
doi: 10.1128/MCB.23.6.2017-2028.2003.

Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipid raft-associated transmembrane adaptor

Dominique Davidson  1 , Marcin Bakinowski, Matthew L Thomas, Vaclav Horejsi, André Veillette
Affiliations

Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipid raft-associated transmembrane adaptor

Dominique Davidson et al. Mol Cell Biol. 2003 Mar.

Abstract

PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.

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Figures

FIG. 1.
FIG. 1.
Effect of TCR stimulation on PAG tyrosine phosphorylation and association with Csk in normal mouse T cells. (A) PAG immunoprecipitations. Mouse thymocytes were stimulated for the indicated periods of time at 37°C with biotinylated anti-CD3 MAb 145-2C11 and avidin. Unstimulated controls were incubated with avidin alone. Unstimulated and stimulated cells were lysed in Brij 58-containing buffer and subjected to sucrose density gradient centrifugation. Lipid raft fractions (fractions 2 and 3) were recovered, solubilized in maltoside, and immunoprecipitated with anti-PAG antibodies. Tyrosine phosphorylation of PAG was monitored by probing of anti-PAG immunoprecipitates with anti-P.tyr antibodies (top panel). The association of PAG with Csk was determined by reprobing the immunoblot membrane with anti-Csk (center panel), while the abundance of PAG was verified by reprobing with anti-PAG (bottom panel). (B) Overall protein tyrosine phosphorylation. Total cell lysates from the experiment depicted in Fig. 1A were probed by anti-P.tyr immunoblotting.
FIG. 2.
FIG. 2.
Overexpression of wild-type PAG and dominant-negative PAG mutants in transgenic mice. (A) Overexpression of PAG in various T-cell populations. Purified T cells from normal control mice or transgenic mice overexpressing wild-type PAG (PAG wt) were probed by immunoblotting of total cell lysates with anti-PAG. Flow cytometry analyses confirmed that >90% of cells in all preparations were T cells (data not shown). Similar results were obtained with transgenic mice expressing PAG Y314F and PAG 9Y→F (data not shown). (B and C) Tyrosine phosphorylation of PAG and its association with Csk. PAG was immunoprecipitated from lipid raft fractions isolated from thymocytes of the indicated mice, and its tyrosine phosphorylation was determined by immunoblotting with anti-P.tyr antibodies (top panels). The association of PAG with Csk was ascertained by reprobing of the immunoblot membrane with anti-Csk (second panels from the top) or by immunoblotting of anti-Csk immunoprecipitates with anti-PAG (third panels from the top). The abundance of PAG (fourth panels from the top) and Csk (fifth panels from the top) was verified by immunoblotting of total cell lysates with anti-PAG and anti-Csk, respectively. Note that in Fig. 2B and C, the duration of the autoradiographic exposures was much shorter than that used for Fig. 1A. This explains the weaker signal of PAG tyrosine phosphorylation (top panels) and PAG-associated Csk (second panels from the top) in control thymocytes. Both immunoreactive products were more clearly seen upon longer autoradiographic exposures (data not shown). The upper band seen in the anti-Csk immunoblots of PAG immunoprecipitates is the heavy chain of immunoglobulin.
FIG. 3.
FIG. 3.
Impact of PAG on antigen receptor-induced proliferation and cytokine production. CD4+ splenic T cells were isolated from the indicated mice and stimulated for 40 to 48 h with medium alone, immobilized anti-CD3 alone (1 or 3 μg/ml), immobilized anti-CD3 (1 or 3 μg/ml) plus soluble anti-CD28 (1 μg/ml), or the combination of PMA (50 ng/ml) plus ionomycin (iono) (100 ng/ml). wt, wild type. (A and B) Thymidine incorporation. All assays were done in triplicate, and average values are shown. (C and D) IL-2 secretion; (E) IL-4 production; (F) IFN-γ production. (G) The experiment was performed as described for Fig. 3A, except that the proliferation assays were in the absence or in the presence of recombinant IL-2 (20 U/ml). For panels C to G, all assays were done in duplicate and average values are shown.
FIG. 4.
FIG. 4.
Regulation of TCR-induced protein tyrosine phosphorylation by PAG. wt, wild type. (A) Overall protein tyrosine phosphorylation. Thymocytes from the indicated mice were stimulated as outlined for Fig. 1, except that biotinylated anti-TCR MAb H57-597 plus avidin was used. Changes in protein tyrosine phosphorylation were monitored by immunoblotting of total cell lysates with anti-P.tyr antibodies. (B) Cell fractionation. Cells were stimulated as described for panel A, except that lysates were fractionated by sucrose density gradient centrifugation. Lysates corresponding to equal cell numbers were obtained from fractions 2 and 3 (lipid raft fractions) or fractions 8 and 9 (soluble fractions) and were probed by immunoblotting with anti-P.tyr (top panel), anti-LAT (center panel), or anti-PAG (bottom panel) antibodies. Total cell lysates were analyzed in lanes 13 to 18.
FIG. 5.
FIG. 5.
Regulation of TCR-induced calcium fluxes by PAG. Thymocytes were loaded with Indo-1 and were stimulated at 37°C with biotinylated anti-TCR MAb H57-597 and avidin. Changes in intracellular calcium were monitored, using a cell sorter, by gating on CD4+ single-positive thymocytes. The ratio of bound Indo-1/free Indo-1 is shown on the ordinate. The arrow corresponds to the moment at which the biotinylated anti-TCR antibody and avidin were present and represents time 0. Cells were observed for 6 min. Similar results were obtained when calcium changes were analyzed in total thymocytes (data not shown). In comparison to normal cells, considerably fewer cells overexpressing wild-type (wt) PAG exhibited a calcium response (20.2% versus 4.6%).
FIG. 6.
FIG. 6.
Impact of constitutively activated Src kinase on PAG-mediated inhibition. Mice overexpressing wild-type PAG were crossed with transgenic mice expressing a constitutively activated version of FynT (FynT Y528F). wt, wild type. (A) Expression of PAG and FynT. Lysates from thymocytes were probed by immunoblotting with anti-PAG (top panel) or anti-Fyn (bottom panel). (B) Thymidine incorporation; (C) IL-2 secretion. Cells were stimulated and assayed as detailed for Fig. 3.
FIG. 7.
FIG. 7.
Cell fractionation experiments. (A and B) Sucrose density gradient centrifugation. The distribution of several molecules in mouse thymocytes was examined by sucrose density gradient centrifugation. Polypeptides were detected by immunoblotting with the indicated antibodies, utilizing aliquots obtained from the various fractions. Lipid rafts corresponded to fractions 2 to 4, while soluble proteins were present in fractions 7 to 9. In the case of GM1 gangliosides, detection was achieved using a dot blot and probing of the membrane with cholera toxin (Tx) coupled to horseradish peroxidase. Similar results were obtained when lysates were prepared with Triton X-100 (data not shown).
FIG. 8.
FIG. 8.
Involvement of PTPs in dephosphorylation of PAG in T cells. (A) Extent of PAG tyrosine phosphorylation in phosphatase-deficient thymocytes. Thymocytes were isolated from the indicated mice, and lysates were fractionated by sucrose density gradient centrifugation. The extent of tyrosine phosphorylation of PAG was evaluated by immunoblotting of fractions corresponding to lipid rafts (fractions 2 to 4) with anti-P.tyr antibodies. (B) PAG tyrosine phosphorylation and association with Csk in CD45-deficient thymocytes. PAG was immunoprecipitated from cell lysates with anti-PAG, and its phosphotyrosine content was examined by anti-P.tyr immunoblotting (top panel). The extent of association of PAG with Csk was determined by immunoblotting of anti-Csk immunoprecipitates with anti-PAG (center panel). The abundance of PAG was verified by immunoblotting of total cell lysates with anti-PAG (bottom panel). (C) TCR-induced dephosphorylation of PAG in phosphatase-deficient thymocytes. Cells were stimulated for 1 min with biotinylated anti-CD3 MAb 145-2C11 and avidin. Unstimulated controls were incubated with avidin alone. Tyrosine phosphorylation of PAG was assessed by probing of anti-PAG immunoprecipitates with anti-P.tyr immunoblotting (top panel). The association with Csk was examined by reprobing the immunoblot membrane with anti-Csk (center panel), whereas the abundance of PAG was verified by reprobing of the immunoblot with anti-PAG (bottom panel). (D) PAG tyrosine phosphorylation and association with Csk in a CD45-negative T-cell line. CD45-positive and CD45-negative derivatives of the mouse T-cell line YAC-1 were examined. PAG was immunoprecipitated from cell lysates with anti-PAG, and its phosphotyrosine content was examined by anti-P.tyr immunoblotting (top panel). The extent of association of PAG with Csk was determined by immunoblotting of anti-PAG immunoprecipitates with anti-Csk (center panel), while the abundance of PAG was verified by immunoblotting of anti-PAG immunoprecipitates with anti-PAG (bottom panel). (E) Overall protein tyrosine phosphorylation in CD45-positive and CD45-negative derivatives of YAC-1. Overall protein tyrosine phosphorylation was determined by immunoblotting of total cell lysates with anti-P.tyr antibodies.
FIG. 9.
FIG. 9.
Substrate-trapping experiment. Cos-1 cells were transiently transfected with the indicated cDNAs, as detailed in the text. (A) Expression levels of the various polypeptides. The abundance of PAG (top panel), ΔSH2 Y505F Lck (center panel) and the two Src-CD45 variants (bottom panel) in total cell lysates was assessed by immunoblotting with the indicated antibodies. (B) Association of PAG with inactive, but not active, CD45. Lysates were immunoprecipitated with the specified antisera and then probed by immunoblotting with the indicated antibodies. NRS, normal rabbit serum.
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References

    1. Blish, C. A., S. R. Dillon, A. G. Farr, and P. J. Fink. 1999. Anergic CD8+ T cells can persist and function in vivo. J. Immunol. 163:155-164. - PubMed
    1. Brdicka, T., D. Pavlistova, A. Leo, E. Bruyns, V. Korinek, P. Angelisova, J. Scherer, A. Shevchenko, A. Shevchenko, I. Hilgert, J. Cerny, K. Drbal, Y. Kuramitsu, B. Kornacker, V. Horejsi, and B. Schraven. 2000. Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation. J. Exp. Med. 191:1591-1604. - PMC - PubMed
    1. Brdickova, N., T. Brdicka, L. Andera, J. Spicka, P. Angelisova, S. L. Milgram, and V. Horejsi. 2001. Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton. FEBS Lett. 507:133-136. - PubMed
    1. Byth, K. F., L. A. Conroy, S. Howlett, A. J. Smith, J. May, D. R. Alexander, and N. Holmes. 1996. CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation. J. Exp. Med. 183:1707-1718. - PMC - PubMed
    1. Cantrell, D. A. 2002. Transgenic analysis of thymocyte signal transduction. Nat. Rev. Immunol. 2:20-27. - PubMed

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