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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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PP2A interacts with CTLA-4 through the lysine rich domain and tyrosine 182. A) Association of CTLA-4 with the A subunit of PP2A. Jurkat T cell lines stably transfected with CTLA-4 variants were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of CTLA-4. Cell lysates were prepared and used for immunoprecipitation of PP2AA, followed by immunoblotting for CTLA-4, PP2AA and PP2AC. B) Association of CTLA-4 with the C subunit of PP2A. Jurkat cells were cultured and lysed as in A) and used for immunoprecipitation of PP2AC, followed by blotting for CTLA-4, PP2AA and PP2AC. Beads, immunoprecipitating Ab without cell lysate. Blots are representative of at least 3 independent experiments.
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Figure 3: PP2A interacts with CTLA-4 through the lysine rich domain and tyrosine 182. A) Association of CTLA-4 with the A subunit of PP2A. Jurkat T cell lines stably transfected with CTLA-4 variants were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of CTLA-4. Cell lysates were prepared and used for immunoprecipitation of PP2AA, followed by immunoblotting for CTLA-4, PP2AA and PP2AC. B) Association of CTLA-4 with the C subunit of PP2A. Jurkat cells were cultured and lysed as in A) and used for immunoprecipitation of PP2AC, followed by blotting for CTLA-4, PP2AA and PP2AC. Beads, immunoprecipitating Ab without cell lysate. Blots are representative of at least 3 independent experiments.

Mentions: PP2A is a heterotrimeric complex comprised of a core dimer consisting of the A and C subunits and a regulatory B subunit thought to provide substrate specificity and/or localization within the cell [11]. Previous reports have demonstrated that CTLA-4 can interact with both the A and C subunits of PP2A [9,10]. Using our panel of CTLA-4 mutants we performed a structural analysis of the association of both PP2AA and PP2AC subunits with CTLA-4. To do this, we induced the expression of the different CTLA-4 mutants, immunoprecipitated PP2A using anti-PP2AA or anti-PP2AC Abs and immunoblotted for CTLA-4 (Figure 3). Under these conditions the amount of immunoprecipitated PP2A represents approximately 1.5% of total PP2A. Equal loading was confirmed by blotting for the immunoprecipitating Ab. As expected, we observed co-precipitation of both PP2AA and PP2AC with WT CTLA-4. On average approximately 2% of total CTLA-4 immunoprecipitated with PP2A. As it is the surface pool of CTLA-4 (representing approximately 10% of total CTLA-4) that associates with PP2A, we estimate that approximately 20% of surface CTLA-4 is associated with PP2A. Additionally, CTLA-4 variants mutated at the proline rich motif were able to co-precipitate with PP2A at similar levels compared to WT CTLA-4, suggesting that these residues are not essential in the CTLA-4:PP2A interaction.


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

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

PP2A interacts with CTLA-4 through the lysine rich domain and tyrosine 182. A) Association of CTLA-4 with the A subunit of PP2A. Jurkat T cell lines stably transfected with CTLA-4 variants were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of CTLA-4. Cell lysates were prepared and used for immunoprecipitation of PP2AA, followed by immunoblotting for CTLA-4, PP2AA and PP2AC. B) Association of CTLA-4 with the C subunit of PP2A. Jurkat cells were cultured and lysed as in A) and used for immunoprecipitation of PP2AC, followed by blotting for CTLA-4, PP2AA and PP2AC. Beads, immunoprecipitating Ab without cell lysate. Blots are representative of at least 3 independent experiments.
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Related In: Results  -  Collection

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Figure 3: PP2A interacts with CTLA-4 through the lysine rich domain and tyrosine 182. A) Association of CTLA-4 with the A subunit of PP2A. Jurkat T cell lines stably transfected with CTLA-4 variants were cultured overnight in the presence of doxycycline (1 μg/ml) to induce the expression of CTLA-4. Cell lysates were prepared and used for immunoprecipitation of PP2AA, followed by immunoblotting for CTLA-4, PP2AA and PP2AC. B) Association of CTLA-4 with the C subunit of PP2A. Jurkat cells were cultured and lysed as in A) and used for immunoprecipitation of PP2AC, followed by blotting for CTLA-4, PP2AA and PP2AC. Beads, immunoprecipitating Ab without cell lysate. Blots are representative of at least 3 independent experiments.
Mentions: PP2A is a heterotrimeric complex comprised of a core dimer consisting of the A and C subunits and a regulatory B subunit thought to provide substrate specificity and/or localization within the cell [11]. Previous reports have demonstrated that CTLA-4 can interact with both the A and C subunits of PP2A [9,10]. Using our panel of CTLA-4 mutants we performed a structural analysis of the association of both PP2AA and PP2AC subunits with CTLA-4. To do this, we induced the expression of the different CTLA-4 mutants, immunoprecipitated PP2A using anti-PP2AA or anti-PP2AC Abs and immunoblotted for CTLA-4 (Figure 3). Under these conditions the amount of immunoprecipitated PP2A represents approximately 1.5% of total PP2A. Equal loading was confirmed by blotting for the immunoprecipitating Ab. As expected, we observed co-precipitation of both PP2AA and PP2AC with WT CTLA-4. On average approximately 2% of total CTLA-4 immunoprecipitated with PP2A. As it is the surface pool of CTLA-4 (representing approximately 10% of total CTLA-4) that associates with PP2A, we estimate that approximately 20% of surface CTLA-4 is associated with PP2A. Additionally, CTLA-4 variants mutated at the proline rich motif were able to co-precipitate with PP2A at similar levels compared to WT CTLA-4, suggesting that these residues are not essential in the CTLA-4:PP2A interaction.

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