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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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Sequence and surface expression CTLA-4 molecules used in these studies. The WT sequence of the intracellular tail of CTLA-4 is shown in full with key residues targeted for mutation depicted in bold. Lysine residues 152, 155 and 156 were changed to alanine residues to generate KLESS CTLA-4. Tyrosine residues located at positions 165 or 182 have been converted to phenylalanine to create Y165F CTLA-4 and Y182F CTLA-4, respectively. The double mutant Y165F/Y182F CTLA-4 contains mutations at both tyrosine residues. PRO- CTLA-4 contains mutations of proline residues 169 and 173 to alanine residues. Stably transfected Jurkat T cell lines have been generated for each of these CTLA-4 variants. Cells were induced overnight with doxycycline (1 μg/ml) and the surface expression of CTLA-4 was measured by flow cytometry (black line, CTLA-4; shaded profile, isotype-matched Ab).
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Figure 2: Sequence and surface expression CTLA-4 molecules used in these studies. The WT sequence of the intracellular tail of CTLA-4 is shown in full with key residues targeted for mutation depicted in bold. Lysine residues 152, 155 and 156 were changed to alanine residues to generate KLESS CTLA-4. Tyrosine residues located at positions 165 or 182 have been converted to phenylalanine to create Y165F CTLA-4 and Y182F CTLA-4, respectively. The double mutant Y165F/Y182F CTLA-4 contains mutations at both tyrosine residues. PRO- CTLA-4 contains mutations of proline residues 169 and 173 to alanine residues. Stably transfected Jurkat T cell lines have been generated for each of these CTLA-4 variants. Cells were induced overnight with doxycycline (1 μg/ml) and the surface expression of CTLA-4 was measured by flow cytometry (black line, CTLA-4; shaded profile, isotype-matched Ab).

Mentions: To dissect the interaction between CTLA-4 and PP2A we used a panel of doxycycline-inducible Jurkat T cells stably transfected with WT CTLA-4 or CTLA-4 molecules containing mutations within the cytoplasmic domain. The intracellular tail of CTLA-4 is 100% conserved among mammalian species suggesting that this domain is important for the function of CTLA-4 [18]. Although the intracellular portion of CTLA-4 does not have any intrinsic enzymatic activity, it contains several motifs that may be important for its interaction with key signaling molecules [4]. Thus, we examined CTLA-4 molecules containing mutations at each of these putative motifs. These included the lysine rich motif (KLESS) located in the juxtamembrane region of the tail and the tyrosine residue located at position 165 (Y165F), both sites previously claimed to be important for the interaction with PP2A [9,10]. CTLA-4 molecules with mutations at the second tyrosine residue (Y182F), at both tyrosine residues (Y165F/Y182F), and at the proline rich domain (PRO-) located at residues 169–173 were also used. These Jurkat T cell lines were cultured overnight with doxycycline to induce the expression CTLA-4. The surface expression of each CTLA-4 variant was measured by flow cytometry (Figure 2). Similar levels of surface expression were observed for cells expressing WT (mean fluorescence intensity (MFI), 691), PRO- (MFI, 330) and Y182F (MFI, 432) CTLA-4 molecules. Lower levels of surface expression were detected for KLESS CTLA-4 (MFI, 43), while an increase in the level of CTLA-4 on the surface was observed for cells expressing molecules mutated at position Y165 (both Y165F (MFI, 1772), and Y165F/Y182F(MFI, 1757)), likely owing to their inability to be effectively internalized by AP-2 [19-21].


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

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

Sequence and surface expression CTLA-4 molecules used in these studies. The WT sequence of the intracellular tail of CTLA-4 is shown in full with key residues targeted for mutation depicted in bold. Lysine residues 152, 155 and 156 were changed to alanine residues to generate KLESS CTLA-4. Tyrosine residues located at positions 165 or 182 have been converted to phenylalanine to create Y165F CTLA-4 and Y182F CTLA-4, respectively. The double mutant Y165F/Y182F CTLA-4 contains mutations at both tyrosine residues. PRO- CTLA-4 contains mutations of proline residues 169 and 173 to alanine residues. Stably transfected Jurkat T cell lines have been generated for each of these CTLA-4 variants. Cells were induced overnight with doxycycline (1 μg/ml) and the surface expression of CTLA-4 was measured by flow cytometry (black line, CTLA-4; shaded profile, isotype-matched Ab).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Sequence and surface expression CTLA-4 molecules used in these studies. The WT sequence of the intracellular tail of CTLA-4 is shown in full with key residues targeted for mutation depicted in bold. Lysine residues 152, 155 and 156 were changed to alanine residues to generate KLESS CTLA-4. Tyrosine residues located at positions 165 or 182 have been converted to phenylalanine to create Y165F CTLA-4 and Y182F CTLA-4, respectively. The double mutant Y165F/Y182F CTLA-4 contains mutations at both tyrosine residues. PRO- CTLA-4 contains mutations of proline residues 169 and 173 to alanine residues. Stably transfected Jurkat T cell lines have been generated for each of these CTLA-4 variants. Cells were induced overnight with doxycycline (1 μg/ml) and the surface expression of CTLA-4 was measured by flow cytometry (black line, CTLA-4; shaded profile, isotype-matched Ab).
Mentions: To dissect the interaction between CTLA-4 and PP2A we used a panel of doxycycline-inducible Jurkat T cells stably transfected with WT CTLA-4 or CTLA-4 molecules containing mutations within the cytoplasmic domain. The intracellular tail of CTLA-4 is 100% conserved among mammalian species suggesting that this domain is important for the function of CTLA-4 [18]. Although the intracellular portion of CTLA-4 does not have any intrinsic enzymatic activity, it contains several motifs that may be important for its interaction with key signaling molecules [4]. Thus, we examined CTLA-4 molecules containing mutations at each of these putative motifs. These included the lysine rich motif (KLESS) located in the juxtamembrane region of the tail and the tyrosine residue located at position 165 (Y165F), both sites previously claimed to be important for the interaction with PP2A [9,10]. CTLA-4 molecules with mutations at the second tyrosine residue (Y182F), at both tyrosine residues (Y165F/Y182F), and at the proline rich domain (PRO-) located at residues 169–173 were also used. These Jurkat T cell lines were cultured overnight with doxycycline to induce the expression CTLA-4. The surface expression of each CTLA-4 variant was measured by flow cytometry (Figure 2). Similar levels of surface expression were observed for cells expressing WT (mean fluorescence intensity (MFI), 691), PRO- (MFI, 330) and Y182F (MFI, 432) CTLA-4 molecules. Lower levels of surface expression were detected for KLESS CTLA-4 (MFI, 43), while an increase in the level of CTLA-4 on the surface was observed for cells expressing molecules mutated at position Y165 (both Y165F (MFI, 1772), and Y165F/Y182F(MFI, 1757)), likely owing to their inability to be effectively internalized by AP-2 [19-21].

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