Different phosphorylation behaviour of regulatory subunit isoforms of type II cAMP-dependent protein kinase from bovine heart.
Two forms of the regulatory subunit of the type II cAMP-dependent protein kinase (RII55 and RII52) were identified from bovine heart by gel electrophoretic behaviour. After autophosphorylation the RII55 isoform migrated more slowly (RII55/57) while the migration of RII52 isoform did not shift. Both isoforms showed different affinity for cAMP. The RII55/57 isoform was eluted from a cAMP-agarose column at 10 mM cAMP at low ionic strength whereas the RII52 isoform required cAMP, plus 2M NaCl. Partial proteolysis, using trypsin or formic acid, of autophosphorylated regulatory subunit isoforms resulted in different cleavage pattern as determined by peptide mapping. However, the V8 125I-peptides patterns of both isoforms are quite similar. Incubation of partially purified holoenzyme with 10 nM [gamma-32P]ATP (low ATP concentration) yielded a single band of Mr = 57,000 which corresponds to the RII55/57 isoform. The incubation, however, at 20 microM [gamma-32P]ATP yielded two phosphobands corresponding to both RII55/57 and RII52 isoforms. The phosphorylation of RII52 took place with a lower efficiency and was more sensitive to the cAMP than the corresponding phosphorylation of the RII55/57.
González-Nicolás
J
J
Centro de Biología Molecular (UAM-CSIC), Universidad Autónoma, Madrid, Spain.
Jiménez
J S
JS
Moreno
F J
FJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D007536:Isomerism; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000235:genetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012697:Serine Endopeptidases
D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase