A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines.
The cAMP protein kinase A (PKA) pathway in T cells conveys an inhibitory signal to suppress inflammation. This study was performed to understand the mechanisms involved in cAMP-mediated signaling in T lymphocytes. A-kinase anchoring proteins (AKAPs) bind and target PKA to various subcellular locations. AKAPs also bind other signaling molecules such as cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cAMP in the cell. PDE4 and PDE7 have important roles in T cell activation. Based on this information, we hypothesized that AKAPs associate with PDEs in T lymphocytes. Immunoprecipitation of Jurkat cell lysates with Abs against both the regulatory subunit of PKA (RIIalpha) and specific AKAPs resulted in increased PDE activity associated with RIIalpha and AKAP95, AKAP149, and myeloid translocation gene (MTG) compared with control (IgG). Immunoprecipitation and pull-down analyses demonstrate that PDE4A binds to AKAP149, AKAP95, and MTG, but not AKAP79, whereas PDE7A was found to bind only MTG. Further analysis of MTG/PDE association illustrated that PDE4A and PDE7A bind residues 1-344 of MTG16b. Confocal analysis of HuT 78 cells stained with anti-PDE7A showed overlapping staining patterns with the Golgi marker GM130, suggesting that PDE7A is located in the Golgi. The staining pattern of PDE7A also showed similarity to the staining pattern of MTG, supporting the immunoprecipitation data and suggesting that MTG may interact with PDE7A in the Golgi. In summary, these data suggest that AKAPs interact with both PKA and PDE in T lymphocytes and thus are a key component of the signaling complex regulating T cell activation.
Veterans Affairs Medical Center, Oregon Health and Sciences University, Portland 97239, USA.
Angela L
AL
Asirvatham
Sarah G
SG
Galligan
Robynn V
RV
Schillace
Michael P
MP
Davey
Valeria
V
Vasta
Joseph A
JA
Beavo
Daniel W
DW
Carr
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics; D013601:T-Lymphocytes / Q000201:enzymology*; D025521:Tumor Suppressor Proteins / Q000235:genetics
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; C485099:AKAP8 protein, human; D048868:Adaptor Proteins, Signal Transducing; C115744:CBFA2T3 protein, human; D002352:Carrier Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases