Functional characterization of cAMP-binding mutations in type I protein kinase

摘要: Abstract A mutant form of the type I regulatory subunit (RI) cAMP-dependent protein kinase has been cloned and sequenced (Clegg, C. H., Correll, L. A., Cadd, G. C., McKnight, S. (1987) J. Biol. Chem. 262, 13111-13119) which contains two point mutations in site B cAMP-binding site, a Gly to Asp at position this report, effect each independent mutation on rate dissociation cAMP from RI, cAMP-mediated activation holoenzyme inducibility cAMP-responsive genes characterized. Dissociation either recombinant wild RI or B1 demonstrated biphasic kinetics, indicating sites with different affinities for cAMP. B2 subunit, however, was monophasic very rapid that had destroyed increased. The constants (Ka) holoenzymes were 40 188 nM, respectively, positive cooperativity, Hill coefficients 1.61 1.67 B1. required much greater concentrations cAMP, 4.7 microM, half-maximal did not display cooperativity. Constitutive expression mouse AtT20 pituitary cells resulted only small shift Ka these compared increased by more than 100-fold. Transient human JEG-3 choriocarcinoma inhibited forskolin promoter 35% whereas similar response 90%. These results suggest amino acid 324 completely blocks binding Arg His 332 causes subtle alteration binding. Expression animal dominant repression activity kinase-mediated processes.