Correction: Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

PKA type Iα, Ezrin, EBP50, Cbp/PAG, and Csk form a supramolecular complex in T cell lipid rafts. A–E, Lipid raft fractions of T lymphocytes were isolated, pooled, and subjected to immunoprecipitation with mouse mAbs against RIα and RIIα (A), Ezrin (B), Csk (C and E), EBP-50 (D), and PKA C (E). Control IgG were included in all immunoprecipitations. Precipitates were analyzed by 10% SDS-PAGE, followed by immunoblotting with indicated Abs. F, Model of multiprotein complex consisting of PKA/Ezrin/EBP50, Csk, and Cbp/PAG.

Knockdown of Ezrin eliminates PKA type I from T cell lipid rafts and disrupts cAMP-mediated inhibition of T cell immune function. Purified T cells were transfected with Ezrin-specific (Ez799, Ez1245) siRNAs or triple G/C-mismatched control siRNAs (Ez799M3, Ez1245M3), immunoblotted to verify Ezrin knockdown, and examined for effect on cAMP-mediated inhibition of IL-2 secretion. A, Effect of Ezrin knockdown on lipid raft localization of Ezrin and PKA RIα. B and D, Ezrin knockdown in one representative experiment corresponding to data shown in C and E. C and E, Effect of Ezrin knockdown on cAMP-mediated inhibition of IL-2 secretion. Forty-eight hours posttransfection, cells were pretreated with 8-CPT-cAMP (0, 10, or 50 μM) and either kept unstimulated or stimulated for 20 h with anti-CD3/anti-CD28-coated beads (bead-to-cell ratio 1:1). Then supernatants were harvested and assessed for IL-2. Levels of IL-2 secretion are shown relative to those of anti-CD3/anti-CD28-stimulated cells. Average ± SEM (n = 6–9) (C) or duplicate measurements (average ± half range) (E) are shown.

Expression of Ht31 competes anchored PKA RIα from lipid rafts and disrupts cAMP inhibition of T cell proliferation. A, Jurkat T cells were transfected with a mammalian expression vector encoding Ht31 and incubated for 16 h at 37°C. T cells were lysed on ice in lysis buffer containing 0.7% Triton X-100 and fractionated on sucrose gradients, as described in Materials and Methods. Fractions were resolved on SDS-PAGE, blotted to PVDF membranes, and probed with mouse mAb against RIα and RIIα. B, Reversal of cAMP-mediated inhibition of TCR/CD3-stimulated T cell proliferation by the use of competitor peptide to compete the TCR/CD3-associated anchoring of RIα subunit of PKA type I in T cells. TCR/CD3-stimulated proliferation of peripheral blood CD3⁺ T cells from normal healthy blood donor following treatment with liposomes alone (mock) or with increasing concentrations (25–100 μM) of a competitor peptide (Ht-31) that competes anchoring to PKA (left panel) or a control peptide (right panel; Ht31-P). Note: reduced sensitivity to cAMP following loading with increasing concentrations of Ht31 competitor peptide, but not with the control peptide (Ht-31P). C, TCR/CD3 stimulated proliferation of peripheral blood CD3⁺ T cells in the presence of increasing concentrations of 8-CPT-cAMP. Normalized levels of proliferation of T cells incubated with liposomes loaded with Ht31-P control peptide (●, continuous line) or Ht31 peptide (35 μM) to compete anchoring of PKA type I (○, dotted line) were assessed as [³H]thymidine incorporation after 48 h during which [³H]thymidine was added for the last 18 h. Note: right shift of the IC50 (arrow) from 1.8 to 4.8 μM in the presence of 8-CPT-cAMP.