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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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The phosphatase activity of PP2A is required for CTLA-4-mediated T cell activation. Stably transfected Jurkat T cells were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of WT CTLA-4 or CTLA-4 mutant molecules. The cells were further cultured in the presence of doxycycline and stimulated with 24:26 (100 μg/ml) in the presence or absence of OA (0.01 μM) for 48 hours at 37°C. Supernatants were harvested and IL-2 production was measured by ELISA. For each CTLA-4 variant the percent of IL-2 produced in the presence of OA was normalized to IL-2 levels in the absence of OA. All graphs are representative of at least two independent experiments.
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Figure 7: The phosphatase activity of PP2A is required for CTLA-4-mediated T cell activation. Stably transfected Jurkat T cells were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of WT CTLA-4 or CTLA-4 mutant molecules. The cells were further cultured in the presence of doxycycline and stimulated with 24:26 (100 μg/ml) in the presence or absence of OA (0.01 μM) for 48 hours at 37°C. Supernatants were harvested and IL-2 production was measured by ELISA. For each CTLA-4 variant the percent of IL-2 produced in the presence of OA was normalized to IL-2 levels in the absence of OA. All graphs are representative of at least two independent experiments.

Mentions: We and others have shown that inhibition of T cell activation by CTLA-4 requires PP2A activity [10,13]. Specifically, CTLA-4 inhibits the activation of Akt, a molecule that is important in many cellular processes including IL-2 production [22]. CTLA-4-mediated inhibition of Akt activity is dependent on the phosphatase activity of PP2A as Akt phosphorylation was shown to be sensitive to the PP2A inhibitor okadaic acid (OA) [13]. Thus, we determined whether the activity of PP2A was important for the inverse agonist properties of CTLA-4. The panel of stably transfected Jurkat T cells was induced overnight in the presence of doxycycline to induce the expression of the CTLA-4 variants. The cells were further cultured with doxycycline and stimulated with 24:26 in the presence or absence of OA. IL-2 production was measured and normalized to the amount of IL-2 produced in the absence of OA for each of the CTLA-4 variants. We found that CTLA-4-mediated T cell activation was significantly inhibited by OA in T cells expressing WT and mutants forms of CTLA-4 excluding KLESS (Figure 7). Inhibition of inverse agonist activation by OA ranged from 45% (for Y165F/Y182F) to 88% (for Y182F) (69% for WT, 70% for PRO-, and 76% for Y165F). As shown above (Figure 5), KLESS CTLA-4 did not respond to 24:26 and thus no effect of OA was apparent (Figure 7). These results suggest that the phosphatase activity of PP2A is required for the inverse agonist response of CTLA-4.


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

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

The phosphatase activity of PP2A is required for CTLA-4-mediated T cell activation. Stably transfected Jurkat T cells were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of WT CTLA-4 or CTLA-4 mutant molecules. The cells were further cultured in the presence of doxycycline and stimulated with 24:26 (100 μg/ml) in the presence or absence of OA (0.01 μM) for 48 hours at 37°C. Supernatants were harvested and IL-2 production was measured by ELISA. For each CTLA-4 variant the percent of IL-2 produced in the presence of OA was normalized to IL-2 levels in the absence of OA. All graphs are 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 7: The phosphatase activity of PP2A is required for CTLA-4-mediated T cell activation. Stably transfected Jurkat T cells were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of WT CTLA-4 or CTLA-4 mutant molecules. The cells were further cultured in the presence of doxycycline and stimulated with 24:26 (100 μg/ml) in the presence or absence of OA (0.01 μM) for 48 hours at 37°C. Supernatants were harvested and IL-2 production was measured by ELISA. For each CTLA-4 variant the percent of IL-2 produced in the presence of OA was normalized to IL-2 levels in the absence of OA. All graphs are representative of at least two independent experiments.
Mentions: We and others have shown that inhibition of T cell activation by CTLA-4 requires PP2A activity [10,13]. Specifically, CTLA-4 inhibits the activation of Akt, a molecule that is important in many cellular processes including IL-2 production [22]. CTLA-4-mediated inhibition of Akt activity is dependent on the phosphatase activity of PP2A as Akt phosphorylation was shown to be sensitive to the PP2A inhibitor okadaic acid (OA) [13]. Thus, we determined whether the activity of PP2A was important for the inverse agonist properties of CTLA-4. The panel of stably transfected Jurkat T cells was induced overnight in the presence of doxycycline to induce the expression of the CTLA-4 variants. The cells were further cultured with doxycycline and stimulated with 24:26 in the presence or absence of OA. IL-2 production was measured and normalized to the amount of IL-2 produced in the absence of OA for each of the CTLA-4 variants. We found that CTLA-4-mediated T cell activation was significantly inhibited by OA in T cells expressing WT and mutants forms of CTLA-4 excluding KLESS (Figure 7). Inhibition of inverse agonist activation by OA ranged from 45% (for Y165F/Y182F) to 88% (for Y182F) (69% for WT, 70% for PRO-, and 76% for Y165F). As shown above (Figure 5), KLESS CTLA-4 did not respond to 24:26 and thus no effect of OA was apparent (Figure 7). These results suggest that the phosphatase activity of PP2A is required for the inverse agonist response of CTLA-4.

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