Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes.
Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.
Schillace
Robynn V
RV
Veterans Affairs Medical Center and Department of Medicine, Oregon Health and Sciences University, Portland, OR 97201, USA.
Andrews
Sarah F
SF
Liberty
Greg A
GA
Davey
Michael P
MP
Carr
Daniel W
DW
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015723:Gene Library; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000166:cytology / Q000201:enzymology / Q000276:immunology*
D002352:Carrier Proteins; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; C517819:PRKAR2A protein, human; D020126:Brefeldin A; D017868:Cyclic AMP-Dependent Protein Kinases