The adaptor protein EBP50 is important for localization of the protein kinase A-Ezrin complex in T-cells and the immunomodulating effect of cAMP.
We recently reported that the dual-specificity AKAP (A-kinaseanchoring protein) Ezrin targets type I PKA (protein kinase A) to the vicinity of the TCR (T-cell receptor) in T-cells and, together with PAG (phosphoprotein associated with glycosphingolipid-enriched membrane microdomains) and EBP50 [ERM (Ezrin/Radixin/Moesin)-binding phosphoprotein 50], forms a scaffold that positions PKA close to its substrate, Csk (C-terminal Src kinase). This complex is important for controlling the activation state of T-cells. Ezrin binds the adaptor protein EBP50, which again contacts PAG. In the present study, we show that Ezrin and EBP50 interact with high affinity (KD=58+/-7 nM). A peptide corresponding to the EB (Ezrin-binding) region in EBP50 (EBP50pep) was used to further characterize the binding kinetics and compete the Ezrin-EBP50 interaction by various methods in vitro. Importantly, loading T-cells with EBP50pep delocalized Ezrin, but not EBP50. Furthermore, disruption of this complex interfered with cAMP modulation of T-cell activation, which is seen as a reversal of cAMP-mediated inhibition of IL-2 (interleukin 2) production, demonstrating an important role of EBP50 in this complex. In summary, both the biochemical and functional data indicate that targeting the Ezrin-EBP interaction could be a novel and potent strategy for immunomodulation.
The Biotechnology Centre of Oslo, University of Oslo, PB 1125 Blindern, N-0317 Oslo, Norway.
Anne Jorunn
AJ
Stokka
Randi
R
Mosenden
Anja
A
Ruppelt
Birgitte
B
Lygren
Kjetil
K
Taskén
D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D056747:Immunomodulation / Q000187:drug effects*; D007376:Interleukin-2; D008213:Lymphocyte Activation; D046912:Multiprotein Complexes; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000502:physiology*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*; D013601:T-Lymphocytes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000099149:Ezrin
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D003598:Cytoskeletal Proteins; D007376:Interleukin-2; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor