Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI alpha 2 C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation.
Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RI alpha, RII alpha, C alpha and C beta, barely detectable levels of RI beta mRNA, and no detectable RII beta or C gamma mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RI alpha and RII alpha, but no RI beta or RII beta. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[32P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RI beta. Immunofluorescence staining of RI alpha and RII alpha demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RI alpha, whereas the RII alpha subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RI alpha subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RII alpha subunits remains unchanged.
Levy
F O
FO
Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Norway.
Rasmussen
A M
AM
Taskén
K
K
Skålhegg
B S
BS
Huitfeldt
H S
HS
Funderud
S
S
Smeland
E B
EB
Hansson
V
V
D001402:B-Lymphocytes / Q000201:enzymology*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D015398:Signal Transduction
D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D046911:Macromolecular Substances; C517834:PRKAR1A protein, human; C517766:PRKAR1B protein, human; C517819:PRKAR2A protein, human; D011947:Receptors, Antigen, B-Cell; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases