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Structure-Function analysis of the CTLA-4 interaction with PP2A.

Teft WA, Chau TA, Madrenas J - BMC Immunol. (2009)

Bottom Line: The tail of CTLA-4 does not have any intrinsic enzymatic activity but is able to associate with several signaling molecules including the serine/threonine phosphatase PP2A.PP2A is a heterotrimeric molecule comprised of a regulatory B subunit associated with a core dimer of a scaffolding (A) and a catalytic (C) subunit.This interaction and the phosphatase activity of PP2A are important for CTLA-4-mediated T cell activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: FOCIS Centre for Clinical Immunology and Immunotherapeutics, Robarts Research Institute, The University of Western Ontario, London, Ontario, Canada. wateft@uwo.ca

ABSTRACT

Background: CTLA-4 functions primarily as an inhibitor of T cell activation. There are several candidate explanations as to how CTLA-4 modulates T cell responses, but the exact mechanism remains undefined. The tail of CTLA-4 does not have any intrinsic enzymatic activity but is able to associate with several signaling molecules including the serine/threonine phosphatase PP2A. PP2A is a heterotrimeric molecule comprised of a regulatory B subunit associated with a core dimer of a scaffolding (A) and a catalytic (C) subunit.

Results: Here, we performed an analysis of the human CTLA-4 interface interacting with PP2A. We show that PP2A interacts with the cytoplasmic tail of CTLA-4 in two different sites, one on the lysine rich motif, and the other on the tyrosine residue located at position 182 (but not the tyrosine 165 of the YVKM motif). Although the interaction between CTLA-4 and PP2A was not required for inhibition of T cell responses, it was important for T cell activation by inverse agonists of CTLA-4. Such an interaction was functionally relevant because the inverse agonists induced IL-2 production in an okadaic acid-dependent manner.

Conclusion: Our studies demonstrate that PP2A interacts with the cytoplasmic tail of human CTLA-4 through two motifs, the lysine rich motif centered at lysine 155 and the tyrosine residue 182. This interaction and the phosphatase activity of PP2A are important for CTLA-4-mediated T cell activation.

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24:26 increases the interaction of CTLA-4 with PP2A. Stably transfected Jurkat T cells were induced overnight with doxycycline (1 μg/ml) to express A) WT CTLA-4, B) KLESS CTLA-4, C) Y165F CTLA-4, D) PRO- CTLA-4, E) Y182F CTLA-4 or F) Y165F/Y182F CTLA-4. The cells were stimulated with or without 24:26 (100 μg/ml) for 60 minutes at 37°C. Cells were washed then lysed in standard lysis buffer containing Triton X-100 (1%). Lysates were used for immunoprecipitation of PP2AA, and subsequently blotted for CTLA-4, PP2AA and PP2AC. All data is representative of at least two independent experiments.
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Figure 6: 24:26 increases the interaction of CTLA-4 with PP2A. Stably transfected Jurkat T cells were induced overnight with doxycycline (1 μg/ml) to express A) WT CTLA-4, B) KLESS CTLA-4, C) Y165F CTLA-4, D) PRO- CTLA-4, E) Y182F CTLA-4 or F) Y165F/Y182F CTLA-4. The cells were stimulated with or without 24:26 (100 μg/ml) for 60 minutes at 37°C. Cells were washed then lysed in standard lysis buffer containing Triton X-100 (1%). Lysates were used for immunoprecipitation of PP2AA, and subsequently blotted for CTLA-4, PP2AA and PP2AC. All data is representative of at least two independent experiments.

Mentions: We have previously reported that 24:26 binding to Y165F CTLA-4 stabilizes the association between PP2A and CTLA-4 [14]. Knowing the CTLA-4 residues involved in the interaction with PP2A allowed us to determine the molecular basis of such an increased association. To do this, we cultured the transfected T cell lines overnight in the presence of doxycycline to induce the expression of each CTLA-4 variant. T cells were then stimulated with 24:26 at 37°C for 60 minutes, lysed, immunoprecipitated with anti-PP2AA Abs and subsequently immunoblotted for CTLA-4. Immunoblotting for PP2AA and PP2AC was used as controls. We observed an increase in the amount of CTLA-4 co-precipitated with PP2AA following 24:26 engagement of WT CTLA-4 (Figure 6A), Y165F CTLA-4 (Figure 6C), and PRO- CTLA-4 (Figure 6D), correlating with the ability to induce T cell activation under similar stimulation conditions. In contrast, ligation of KLESS CTLA-4 with 24:26 was unable to induce co-precipitation with PP2AA (Figure 6B). Although little or no association was detected between PP2AA and Y182F CTLA-4 or Y165F/Y182F CTLA-4 in unstimulated conditions, a small increase in the level of association was seen upon inverse agonist ligation of these CTLA-4 molecules (Figure 6E, F). This low level of association correlated with the ability of Y182F CTLA-4 and Y165F/Y182F CTLA-4 to induce some IL-2 production when engaged with the inverse agonist 24:26, respectively.


Structure-Function analysis of the CTLA-4 interaction with PP2A.

Teft WA, Chau TA, Madrenas J - BMC Immunol. (2009)

24:26 increases the interaction of CTLA-4 with PP2A. Stably transfected Jurkat T cells were induced overnight with doxycycline (1 μg/ml) to express A) WT CTLA-4, B) KLESS CTLA-4, C) Y165F CTLA-4, D) PRO- CTLA-4, E) Y182F CTLA-4 or F) Y165F/Y182F CTLA-4. The cells were stimulated with or without 24:26 (100 μg/ml) for 60 minutes at 37°C. Cells were washed then lysed in standard lysis buffer containing Triton X-100 (1%). Lysates were used for immunoprecipitation of PP2AA, and subsequently blotted for CTLA-4, PP2AA and PP2AC. All data is representative of at least two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683795&req=5

Figure 6: 24:26 increases the interaction of CTLA-4 with PP2A. Stably transfected Jurkat T cells were induced overnight with doxycycline (1 μg/ml) to express A) WT CTLA-4, B) KLESS CTLA-4, C) Y165F CTLA-4, D) PRO- CTLA-4, E) Y182F CTLA-4 or F) Y165F/Y182F CTLA-4. The cells were stimulated with or without 24:26 (100 μg/ml) for 60 minutes at 37°C. Cells were washed then lysed in standard lysis buffer containing Triton X-100 (1%). Lysates were used for immunoprecipitation of PP2AA, and subsequently blotted for CTLA-4, PP2AA and PP2AC. All data is representative of at least two independent experiments.
Mentions: We have previously reported that 24:26 binding to Y165F CTLA-4 stabilizes the association between PP2A and CTLA-4 [14]. Knowing the CTLA-4 residues involved in the interaction with PP2A allowed us to determine the molecular basis of such an increased association. To do this, we cultured the transfected T cell lines overnight in the presence of doxycycline to induce the expression of each CTLA-4 variant. T cells were then stimulated with 24:26 at 37°C for 60 minutes, lysed, immunoprecipitated with anti-PP2AA Abs and subsequently immunoblotted for CTLA-4. Immunoblotting for PP2AA and PP2AC was used as controls. We observed an increase in the amount of CTLA-4 co-precipitated with PP2AA following 24:26 engagement of WT CTLA-4 (Figure 6A), Y165F CTLA-4 (Figure 6C), and PRO- CTLA-4 (Figure 6D), correlating with the ability to induce T cell activation under similar stimulation conditions. In contrast, ligation of KLESS CTLA-4 with 24:26 was unable to induce co-precipitation with PP2AA (Figure 6B). Although little or no association was detected between PP2AA and Y182F CTLA-4 or Y165F/Y182F CTLA-4 in unstimulated conditions, a small increase in the level of association was seen upon inverse agonist ligation of these CTLA-4 molecules (Figure 6E, F). This low level of association correlated with the ability of Y182F CTLA-4 and Y165F/Y182F CTLA-4 to induce some IL-2 production when engaged with the inverse agonist 24:26, respectively.

Bottom Line: The tail of CTLA-4 does not have any intrinsic enzymatic activity but is able to associate with several signaling molecules including the serine/threonine phosphatase PP2A.PP2A is a heterotrimeric molecule comprised of a regulatory B subunit associated with a core dimer of a scaffolding (A) and a catalytic (C) subunit.This interaction and the phosphatase activity of PP2A are important for CTLA-4-mediated T cell activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: FOCIS Centre for Clinical Immunology and Immunotherapeutics, Robarts Research Institute, The University of Western Ontario, London, Ontario, Canada. wateft@uwo.ca

ABSTRACT

Background: CTLA-4 functions primarily as an inhibitor of T cell activation. There are several candidate explanations as to how CTLA-4 modulates T cell responses, but the exact mechanism remains undefined. The tail of CTLA-4 does not have any intrinsic enzymatic activity but is able to associate with several signaling molecules including the serine/threonine phosphatase PP2A. PP2A is a heterotrimeric molecule comprised of a regulatory B subunit associated with a core dimer of a scaffolding (A) and a catalytic (C) subunit.

Results: Here, we performed an analysis of the human CTLA-4 interface interacting with PP2A. We show that PP2A interacts with the cytoplasmic tail of CTLA-4 in two different sites, one on the lysine rich motif, and the other on the tyrosine residue located at position 182 (but not the tyrosine 165 of the YVKM motif). Although the interaction between CTLA-4 and PP2A was not required for inhibition of T cell responses, it was important for T cell activation by inverse agonists of CTLA-4. Such an interaction was functionally relevant because the inverse agonists induced IL-2 production in an okadaic acid-dependent manner.

Conclusion: Our studies demonstrate that PP2A interacts with the cytoplasmic tail of human CTLA-4 through two motifs, the lysine rich motif centered at lysine 155 and the tyrosine residue 182. This interaction and the phosphatase activity of PP2A are important for CTLA-4-mediated T cell activation.

Show MeSH
Related in: MedlinePlus