Biochem. J. (2010) 425
(381–388) (Printed in Great Britain)
The adaptor protein EBP50 is important for localization of the protein kinase A–Ezrin complex in T-cells and the immunomodulating effect of cAMP
Anne Jorunn Stokka*†, Randi Mosenden*†, Anja Ruppelt*†1, Birgitte Lygren*† and Kjetil Taskén*†2
*The Biotechnology Centre of Oslo, University of Oslo, PB 1125 Blindern, N-0317 Oslo, Norway, and †Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, PB 1125 Blindern, N-0317 Oslo, Norway
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.
Key words: Ezrin, Ezrin/Radixin/Moesin (ERM)-binding phosphoprotein 50 (EBP50), immunomodulation, peptide disruption, protein kinase A (PKA), T-cell receptor (TCR).
Abbreviations used: AKAP, A-kinase-anchoring protein; AlphaScreen, amplified luminescence proximity homogeneous assay screen; b, biotin-labelled; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; Csk, C-terminal Src kinase; DTT, dithiothreitol; EB domain, Ezrin-binding domain; ERM, Ezrin/Radixin/Moesin; EBP50, ERM-binding phosphoprotein 50; FERM domain, N-terminal 4.1 ERM domain; FP, fluorescence polarization; GST, glutathione transferase; IL-2 interleukin 2; IS, immunological synapse; PAG, phosphoprotein associated with glycosphingolipid-enriched membrane microdomains; PKA, protein kinase A; RI, type I regulatory subunit; SMC, small molecular compound; SPR, surface plasmon resonance; TCR, T-cell receptor.
1Present address: Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.
2To whom correspondence should be addressed (email kjetil.tasken@biotek.uio.no).
Received 27 July 2009/5 October 2009; accepted 26 October 2009
Published as BJ Immediate Publication 26 October 2009, doi:10.1042/BJ20091136
© The Authors Journal compilation © 2010 Biochemical Society
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