TAILIEUCHUNG - Báo cáo khoa học: 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 immunoprecipita-tion 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 Ca1 (40 kDa) and Cb2 (47 kDa) in these cells. | ềFEBS Journal Identification and characterization of novel PKA holoenzymes in human T lymphocytes Sigurd 0rstavik1 Ane Funderud1 Tilahun Tolesa Hafte1 Sissel Eikvar1 2 Tore Jahnsen2 and Bj0rn Steen Skalhegg1 1 Department of Nutrition Institute of Basic MedicalSciences Faculty of Medicine University of Oslo Norway 2 Department of Biochemistry Institute of Basic MedicalSciences Faculty of Medicine University of Oslo Norway Keywords antibodies cAMP lymphocytes protein kinase A Correspondence B. Steen Skalhegg Department of Nutrition Institute of Basic MedicalSciences Faculty of Medicine University of Oslo PO Box 1046 Blindern N-0316 Oslo Norway Fax 47 2285 1347 Tel 47 2285 1547 E-mail Website http Received 2 November 2004 revised 22 December 2004 accepted 13 January 2005 doi 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 Ca1 40 kDa and CP2 47 kDa in these cells. Anti-RIa and Anti-RIIa immunoprecipitations demonstrated that both Ca1 and CP2 associate with RIa and RIIa to form PKAI and PKAII holoenzymes. Moreover Anti-CP2 immunoprecipitation revealed that Ca1 coimmunoprecipitates with CP2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme released Ca1 but not CP2 from the Anti-CP2 precipitate indicating that CP2 and Ca1 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. In the absence of cAMP cAMP-dependent protein kinase PKA is a holoenzyme consisting of two regulatory

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