Different phosphorylation behaviour of regulatory subunit isoforms of type 11 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 (RII_55/57) while the migration of RII₅₂ isoform did not shift. Both isoforms showed different affinity for cAMP. The RII_55/57 isoform was eluted from a cAMP-agarose column at 10 mM cAMP at low ionic strenght whereas the RII₅₂ isoform required cAMP, plus 2 M 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 V8¹²⁵I-peptides patterns of both isoforms are quite similar.Incubation of partially purified holoenzyme with 10 nM -³²P]ATP (low ATP concentration) yielded a single band of Mr = 57,000 which corresponds to the RII_55/57 isoform. The incubation, however, at 20 µM -³²P]ATP yielded two phosphobands corresponding to both RII_55/57 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 RII_55/57.