Characterization of in-vitro-translated human regulatory and catalytic subunits of cAMP-dependent protein kinases.
Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all the human R (RI alpha, RI beta, RII alpha and RII beta) and C (C alpha, C beta and C gamma) subunits in a wheat-germ extract. [35S]Methionine-labelled in-vitro-translated products were analyzed by SDS/PAGE and revealed distinct protein bands with apparent molecular masses of 49 (RI alpha), 54-55 (RI beta), 51 (RII alpha) and 53 kDa (RII beta) for the R subunits. In vitro transcription/translation of the cDNAs for the C subunits of PKA gave proteins with molecular masses of approximately 40 kDa for all the different C subunits. Phosphorylation of RII alpha and RII beta by the C subunit of PKA, revealed a distinct mobility shift of the RII alpha subunit on one-dimensional SDS/PAGE (51-54 kDa), but not of RII beta (53 kDa). Further characterization of the R subunits by two-dimensional SDS/PAGE revealed that RI alpha was more acidic than RI beta, with pIs of 6.1-6.0 and 6.4-6.2, respectively. Furthermore, the RII alpha protein was more basic than RII beta, with pIs of approximately 5.4-5.3 and 5.3-5.1, respectively. All the in-vitro-translated R subunits could be photoaffinity labelled by the cAMP-analog 8-azido-[32P]cAMP and were also detected by immunoprecipitation with subunit-specific antibodies.
Foss
K B
KB
Institute of Medical Biochemistry, University of Oslo, Norway.
Landmark
B
B
Skålhegg
B S
BS
Taskén
K
K
Jellum
E
E
Hansson
V
V
Jahnsen
T
T
D000345:Affinity Labels; D002474:Cell-Free System; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008970:Molecular Weight; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D014158:Transcription, Genetic
D000345:Affinity Labels; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D018076:DNA, Complementary; C517834:PRKAR1A protein, human; C517766:PRKAR1B protein, human; C517819:PRKAR2A protein, human; C517837:PRKAR2B protein, human; D017868:Cyclic AMP-Dependent Protein Kinases