Identification and characterization of novel PKA holoenzymes in human T lymphocytes.
Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.
Orstavik
Sigurd
S
Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Norway.
Funderud
Ane
A
Hafte
Tilahun Tolesa
TT
Eikvar
Sissel
S
Jahnsen
Tore
T
Skålhegg
Bjørn Steen
BS
D000906:Antibodies / Q000276:immunology; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D020035:Holoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology
D000906:Antibodies; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D020035:Holoenzymes; C517834:PRKAR1A protein, human; C517819:PRKAR2A protein, human; D017868:Cyclic AMP-Dependent Protein Kinases