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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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Inhibitory function of CTLA-4 molecules with mutation in the cytoplasmic domain. A) Jurkat T cells stably transfected with WT CTLA-4 or mutant CTLA-4 constructs were cultured overnight in the absence or presence of doxycycline (1 μg/ml). Cells were further stimulated with APCs and SEE (1 and 10 ng/ml) for 24 hours at 37°C. IL-2 production was measured by ELISA. Inhibition of IL-2 was determined for each CTLA-4 variant by calculating the percent of IL-2 produced in the presence of CTLA-4 expression normalized to the maximal IL-2 level in the absence of CTLA-4 at each concentration of SEE. The maximal level of inhibition was plotted for each CTLA-4 variant. This graph was generated using triplicate data for each concentration of SEE, from three independent experiments. B) CD28 expression is required for CTLA-4 mediated inhibition. CD28+, CD28- and CD28-reconstituted Jurkat T cells were cultured overnight without (black squares) or with doxycycline (1 μg/ml) (black triangles) to induce the expression of Y165F CTLA-4. Cells were stimulated with SEE:APC and IL-2 was measured as in A). **, p < 0.01; ***, p < 0.001.
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Figure 4: Inhibitory function of CTLA-4 molecules with mutation in the cytoplasmic domain. A) Jurkat T cells stably transfected with WT CTLA-4 or mutant CTLA-4 constructs were cultured overnight in the absence or presence of doxycycline (1 μg/ml). Cells were further stimulated with APCs and SEE (1 and 10 ng/ml) for 24 hours at 37°C. IL-2 production was measured by ELISA. Inhibition of IL-2 was determined for each CTLA-4 variant by calculating the percent of IL-2 produced in the presence of CTLA-4 expression normalized to the maximal IL-2 level in the absence of CTLA-4 at each concentration of SEE. The maximal level of inhibition was plotted for each CTLA-4 variant. This graph was generated using triplicate data for each concentration of SEE, from three independent experiments. B) CD28 expression is required for CTLA-4 mediated inhibition. CD28+, CD28- and CD28-reconstituted Jurkat T cells were cultured overnight without (black squares) or with doxycycline (1 μg/ml) (black triangles) to induce the expression of Y165F CTLA-4. Cells were stimulated with SEE:APC and IL-2 was measured as in A). **, p < 0.01; ***, p < 0.001.

Mentions: We have previously shown that PP2A plays a role as a negative regulator of the inhibitory function of CTLA-4, its primary function in vivo [10]. Therefore, we next determined the ability of each of the CTLA-4 variants examined above to attenuate T cell responses against the staphylococcal enterotoxin E (SEE) superantigen. Jurkat T cell lines containing the mutant CTLA-4 constructs were cultured overnight with or without doxycycline to induce the expression of CTLA-4 and were then stimulated in the presence of APC expressing B7 molecules on their surface and SEE at the indicated concentrations at 37°C for 24 hours (Figure 4A). The maximal level of inhibition was determined for each CTLA-4 variant by calculating the percent of IL-2 produced in the presence of CTLA-4 expression compared to cells stimulated in the absence of CTLA-4 induction. WT CTLA-4 as well as each mutant CTLA-4 was functionally able to inhibit T cell responses (Figure 4A). Cells expressing WT CTLA-4 attenuated IL-2 production in response to SEE stimulation by 65% on average. IL-2 production was inhibited between 68–78% by cells expressing CTLA-4 molecules with mutations in the lysine rich domain and the tyrosine 165 residue (KLESS, Y165F and Y165F/Y182F CTLA-4), while PRO- and Y182F CTLA-4 variants abrogated the response by 41% and 46%, respectively. The ability of each CTLA-4 variant to inhibit T cell responses suggests that the interaction between CTLA-4 and PP2A is not in itself the determinant of the inhibitory function of CTLA-4 and that there are additional factors that play a role in mediating this function.


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

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

Inhibitory function of CTLA-4 molecules with mutation in the cytoplasmic domain. A) Jurkat T cells stably transfected with WT CTLA-4 or mutant CTLA-4 constructs were cultured overnight in the absence or presence of doxycycline (1 μg/ml). Cells were further stimulated with APCs and SEE (1 and 10 ng/ml) for 24 hours at 37°C. IL-2 production was measured by ELISA. Inhibition of IL-2 was determined for each CTLA-4 variant by calculating the percent of IL-2 produced in the presence of CTLA-4 expression normalized to the maximal IL-2 level in the absence of CTLA-4 at each concentration of SEE. The maximal level of inhibition was plotted for each CTLA-4 variant. This graph was generated using triplicate data for each concentration of SEE, from three independent experiments. B) CD28 expression is required for CTLA-4 mediated inhibition. CD28+, CD28- and CD28-reconstituted Jurkat T cells were cultured overnight without (black squares) or with doxycycline (1 μg/ml) (black triangles) to induce the expression of Y165F CTLA-4. Cells were stimulated with SEE:APC and IL-2 was measured as in A). **, p < 0.01; ***, p < 0.001.
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Figure 4: Inhibitory function of CTLA-4 molecules with mutation in the cytoplasmic domain. A) Jurkat T cells stably transfected with WT CTLA-4 or mutant CTLA-4 constructs were cultured overnight in the absence or presence of doxycycline (1 μg/ml). Cells were further stimulated with APCs and SEE (1 and 10 ng/ml) for 24 hours at 37°C. IL-2 production was measured by ELISA. Inhibition of IL-2 was determined for each CTLA-4 variant by calculating the percent of IL-2 produced in the presence of CTLA-4 expression normalized to the maximal IL-2 level in the absence of CTLA-4 at each concentration of SEE. The maximal level of inhibition was plotted for each CTLA-4 variant. This graph was generated using triplicate data for each concentration of SEE, from three independent experiments. B) CD28 expression is required for CTLA-4 mediated inhibition. CD28+, CD28- and CD28-reconstituted Jurkat T cells were cultured overnight without (black squares) or with doxycycline (1 μg/ml) (black triangles) to induce the expression of Y165F CTLA-4. Cells were stimulated with SEE:APC and IL-2 was measured as in A). **, p < 0.01; ***, p < 0.001.
Mentions: We have previously shown that PP2A plays a role as a negative regulator of the inhibitory function of CTLA-4, its primary function in vivo [10]. Therefore, we next determined the ability of each of the CTLA-4 variants examined above to attenuate T cell responses against the staphylococcal enterotoxin E (SEE) superantigen. Jurkat T cell lines containing the mutant CTLA-4 constructs were cultured overnight with or without doxycycline to induce the expression of CTLA-4 and were then stimulated in the presence of APC expressing B7 molecules on their surface and SEE at the indicated concentrations at 37°C for 24 hours (Figure 4A). The maximal level of inhibition was determined for each CTLA-4 variant by calculating the percent of IL-2 produced in the presence of CTLA-4 expression compared to cells stimulated in the absence of CTLA-4 induction. WT CTLA-4 as well as each mutant CTLA-4 was functionally able to inhibit T cell responses (Figure 4A). Cells expressing WT CTLA-4 attenuated IL-2 production in response to SEE stimulation by 65% on average. IL-2 production was inhibited between 68–78% by cells expressing CTLA-4 molecules with mutations in the lysine rich domain and the tyrosine 165 residue (KLESS, Y165F and Y165F/Y182F CTLA-4), while PRO- and Y182F CTLA-4 variants abrogated the response by 41% and 46%, respectively. The ability of each CTLA-4 variant to inhibit T cell responses suggests that the interaction between CTLA-4 and PP2A is not in itself the determinant of the inhibitory function of CTLA-4 and that there are additional factors that play a role in mediating this function.

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