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.
cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.
Ruppelt
Anja
A
The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
Mosenden
Randi
R
Grönholm
Mikaela
M
Aandahl
Einar M
EM
Tobin
Derek
D
Carlson
Cathrine R
CR
Abrahamsen
Hilde
H
Herberg
Friedrich W
FW
Carpén
Olli
O
Taskén
Kjetil
K
D054758:A Kinase Anchor Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000199:Actins / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology*; D054743:Cyclic AMP-Dependent Protein Kinase Type I / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D003599:Cytoskeleton / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000378:metabolism / Q000502:physiology*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000502:physiology; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000494:pharmacology; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D000099149:Ezrin
D054758:A Kinase Anchor Proteins; D000199:Actins; D000242:Cyclic AMP; D054743:Cyclic AMP-Dependent Protein Kinase Type I; D003598:Cytoskeletal Proteins; D007166:Immunosuppressive Agents; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor