cAMP-dependent Protein Kinase Regulatory Subunit Type IIβ

Abstract

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIβ and two mutant RIIβ subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding a protein contains cAMP-binding and and by and The RIIβ and and The Arg mutant the conserved Arg binding and of each and binding to A The of the A mutant protein of and the to the to The mutant the A the of the a to the of the of the a protein to the cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIβ and two mutant RIIβ subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding a protein contains cAMP-binding and and by and The RIIβ and and The Arg mutant the conserved Arg binding and of each and binding to A The of the A mutant protein of and the to the to The mutant the A the of the a to the of the of the a protein to the in and to cAMP-dependent protein kinase cAMP-dependent protein A cAMP-binding of and cAMP-binding catalytic regulatory cAMP-dependent protein A cAMP-binding of and cAMP-binding catalytic regulatory in and by regulatory (R) subunit and in and and a conserved and of two cAMP-binding the A and and a the the catalytic (C) subunit A in the A of and RIIβ The of the by the sequential binding of to the by a binding to the A and the of active and the A and and the of the the conserved of cAMP-binding the The in and a the of the by The contains the of a conserved the Arg each cAMP-binding to and the by to and to a the Arg and the and of and The conserved Arg of each to a in the of the cAMP-binding of Arg in the by to to of to The of to the in the cAMP-binding and the conserved Arg of the conserved Arg to Lys in cAMP-binding of binding to the by the the conserved Arg to Lys in to the of the of the the two binding domains, and of the a to the the by the A to describe here the of the conserved Arg to Lys in the A and of in RIIβ in a RIIβ the in and and the B. The R subunit of of RIIβ to a and a and of B. of the the of to in two the of each conserved Arg and the of each cAMP-binding to the and of the each conserved Arg in RIIβ in the A and in the to the conserved in the of each cAMP-binding and the and the two cAMP-binding The the cAMP-binding and the by a in and the RIIβ subunit the in the A in the The by the and of RIIβ and a mutant protein the The two Arg mutant RIIβ by a the mutant in The of the the of the to a of the the and the The protein in of monomer by by protein by of and The the of the a of the RIIβ of in in designated of and by in the of the mutant The mutant protein to Each protein and a and the the of the R the and and the of R the and of each in the of the the of and protein to the the of The the protein of by the and to the each protein the by the and in a of by of of the of and by a in the to of to the the mutant of the and and mutant RIIβ a The to a a binding in of the the by of of and in the the the by and of Each by the of and of the conserved Arg to Lys mutant by of to the dimer bound by a protein by the Arg mutant the mutant of to of the binding in the of bound of of and the and mutant RIIβ of bound the mutant binding and RIIβ to the of RIIβ a of the The of each protein in the the and the of the The of the in the to the of of and to a of the of and of to the the RIIβ contains two in the and in cAMP-binding the of RIIβ to of the and mutant the in the A The the the the of the The of the by the to in and the and in and The to by the the of of in RIIβ by of in the and by of in to bound to the A and in the A bound to of RIIβ and of the RIIβ to to the of the mutant to the protein and of the to in the A the the A in by RIIβ cAMP-binding in the and of each a in A in the of the A of the the by Each protein designated and of the by the in of of and mutant in a of a and of The of a to to of of The in the the the of and binding the of the A of RIIβ of the conserved in RIIβ the protein and RIIβ of each of the of binding of by binding to the by and by the of the the and the in of the binding to the of the conserved Arg The of the binding and the and mutant a and the and mutant the binding to RIIβ of to binding the and the the in the and of the a of the protein a to the the mutant protein the of RIIβ the the mutant protein a in and a in a in the of the to the of RIIβ the and RIIβ and binding The and binding of the of the Arg mutant and of by of and a of of and of binding and and RIIβ Arg mutant in a of a in the to in the The and the mutant to the of the the The of the A of of the the A the the A the the and to the to the RIIβ the the mutant a in to in the the and of the binding bound to the A mutant to the the the by of by by the the and the The of the protein and the to the the mutant protein a in the the and the of binding to the A the the A to the A to the the the protein the of the of the protein binding to the of mutant by binding to the A to the of the to to A in the mutant of the the of the RIIβ of the the RIIβ the The of in the mutant the protein of the in mutant the the binding to the of the the the a the of cAMP-binding in the of to a in to the of the two binding a each and the two the protein two the of the to the in the the to protein and in the of cAMP-binding A and binding to of conserved to the binding of each and the two in the of the cAMP-binding of the conserved binding the of each and the two The in the the of A in a the and the of the A to a the and RIIβ the of the to a in the cAMP-binding of by a the and B. of the of the of the of of a of by of of a in the of RIIβ to of the in the A the by the in the of the A to RIIβ of the the two and the of the conserved the of the of of to to the A the A the to A the in the the RIIβ to the binding to the A to the A of the mutant protein to the and the of the the and the the of the mutant to the the of A and to of the mutant a in the the and RIIβ The A mutant to the RIIβ bound to the the of the RIIβ bound to the in of the A to binding of the A of the mutant protein the and RIIβ the to the properties the A to the the the of the RIIβ mutant protein and a the binding to the and the of the of the RIIβ to the RIIβ The the in the of the in the the the and of the to the the The and to of to the of and RIIβ the A and in the A and the of the and RIIβ cAMP-binding the of the in the of the and and a each the of the A to the to the by a the of the of the by the the of and RIIβ the of the to the A The by binding in and by the each the and a the to of RIIβ the binding to the A in the of a the and the and of the A to the of the binding in RIIβ the A the of the conserved in and RIIβ and Arg in the of the the to the in a in the of the A to the binding the to cAMP-binding A in the to of the two and in a the of the A to the cAMP-binding in in and RIIβ in and and in and in The binding the the the and the A and domains, and the of the the in to to binding and to the in the cAMP-binding to in in the A and in the in RIIβ cAMP-binding the in the to of the of the cAMP-binding domains, of the A binding by cAMP-binding in by the in The and in the A to the of the The conserved to the Arg by in and the in and RIIβ a of the and RIIβ a conserved the and RIIβ a of the two cAMP-binding and of each the in We the a the the and the the the cAMP-binding and in a of and of the of in and to cAMP-dependent protein kinase cAMP-dependent protein A cAMP-binding of and cAMP-binding catalytic regulatory cAMP-dependent protein A cAMP-binding of and cAMP-binding catalytic regulatory in and by regulatory (R) subunit and in and and a conserved and of two cAMP-binding the A and and a the the catalytic (C) subunit A in the A of and RIIβ The of the by the sequential binding of to the by a binding to the A and the of active and the A and and the of the the conserved of cAMP-binding the The in and a the of the by The contains the of a conserved the Arg each cAMP-binding to and the by to and to a the Arg and the and of and The conserved Arg of each to a in the of the cAMP-binding The of Arg in the by to to of to The of to the in the cAMP-binding and the conserved Arg of the conserved Arg to Lys in cAMP-binding of binding to the by the the conserved Arg to Lys in to the of the of the the two binding domains, and of the a to the the by the A to We describe here the of the conserved Arg to Lys in the A and of in RIIβ in a RIIβ the in and and the B. The R subunit of of RIIβ to a and a and of B. of the the of to in two the of each conserved Arg and the of each cAMP-binding to the and of the each conserved Arg in RIIβ in the A and in the to the conserved in the of each cAMP-binding and the and the two cAMP-binding The the cAMP-binding and the by a in and the RIIβ subunit the in the A in the The by the and of RIIβ and a mutant protein the The two Arg mutant RIIβ by a the mutant in The of the the of the to a of the the and the The protein in of monomer by by protein by of and The the of the a of the RIIβ of in in designated of and by in the of the mutant The mutant protein to Each protein and a and the the of the R the and and the of R the and of each in the of the the of and protein to the the of The the protein of by the and to the each protein the by the and in a of by of of the of and by a in the to of to the the mutant of the and and mutant RIIβ a The to a a binding in of the the by of of and in the the the by and of Each by the of and the RIIβ subunit the in the A in the The by the and of RIIβ and a mutant protein the The two Arg mutant RIIβ by a the mutant in The of the the of the to a of the the and the The protein in of monomer by by protein by of and The the of the a of the RIIβ of in in designated of and by in the of the mutant The mutant protein to Each protein and a and the the of the R the and and the of R the and of each in the of the the of and protein to the the of The the protein of by the The and to the each protein the by the and in a of by of of the of and by a in the to of to the the mutant of the and and mutant RIIβ a The to a a binding in of the the by of of and in the the the by and of Each by the of and of the conserved Arg to Lys mutant by of to the dimer bound by a protein by the Arg mutant the mutant of to of the binding in the of bound of of and the and mutant RIIβ of bound the mutant binding and RIIβ to the of RIIβ a of the The of each protein in the the and the of the The of the in the to the of to the the RIIβ contains two in the and in cAMP-binding the of RIIβ to of the and mutant the in the A The the the the of the The of the by the to in and the and in and The to by the the of of in RIIβ by of in the and by of in to bound to the A and in the A bound to of RIIβ and of the RIIβ to to the of the mutant to the protein and of the to in the A the the A in by RIIβ cAMP-binding in the and of each a in A in the of the A of the the by Each protein designated and of the by the in of of and mutant in a of a and of The of a to to of of The in the the the of and binding the of the A of RIIβ of the conserved in RIIβ the protein and RIIβ of each of the of binding of by binding to the by and by the of the the and the in of the binding to the of the conserved Arg The of the binding and the and mutant a and the and mutant the binding to RIIβ of to binding the and the the in the and of the a of the protein a to the the mutant protein the of RIIβ the the mutant protein a in and a in a in the of the to the of RIIβ the and RIIβ and binding The and binding of the of the Arg mutant and of by of and a of of and of binding and and RIIβ Arg mutant in a of a in the to in the The and the mutant to the of the the The of the A of of the the A the the A the the and to the to the RIIβ the the mutant a in to in the the and of the binding bound to the A mutant to the the the by of by by the the and the The of the protein and the to the the mutant protein a in the the and the of binding to the A the the A to the A to the the the protein the of the of the protein binding to the of mutant by binding to the A to the of the to to A in the mutant of the the of the RIIβ of the the RIIβ the The of in the mutant the protein of the in mutant the the binding to the of the the the a the of and of the conserved Arg to Lys mutant by of to the dimer bound by a protein by the Arg mutant the mutant of to of the binding in the of bound of of and the and mutant RIIβ of bound the mutant binding The conserved Arg to Lys mutant by of to the dimer bound by a protein by the Arg mutant the mutant of to The of the binding in the of bound of of and the and mutant RIIβ of bound the mutant binding and RIIβ to the of RIIβ a of the The of each protein in the the and the of the The of the in the to the of to the the RIIβ contains two in the and in cAMP-binding the of RIIβ to of the and mutant the in the A The the the the of the The of the by the to in and the and in and The to by the the of of in RIIβ by of in the and by of in to bound to the A and in the A bound to B. to the of RIIβ a of the The of each protein in the the and the of the The of the in the to the of to the the RIIβ contains two in the and in cAMP-binding the of RIIβ to of the and mutant the in the A The the the the of the The of the by the to in and the and in and The to by the the of of in RIIβ by of in the and by of in to bound to the A and in the A bound to B. of RIIβ and of the RIIβ to to the of the mutant to the protein and of the to in the A the the A in by RIIβ cAMP-binding in the and of each a in A in the of the A of the of and mutant in a of a and of The of a to to of of The in the the the of and binding the of the A The of the RIIβ to to the of the mutant to the protein and of the to in the A the the A in by RIIβ cAMP-binding in the and of each a in A in the of the A of the of a and of The of a to to of of The in the the the of and binding the of the A of RIIβ of the conserved in RIIβ the protein and RIIβ of each of the of binding of by binding to the by and by the of the the and the in of the binding to the of the conserved Arg The of the binding and the and mutant a and the and mutant the binding to RIIβ of to binding the and the the in the and of the a of the protein a to the the mutant protein the of RIIβ the the mutant protein a in and a in a in the of the to the of RIIβ the of binding and and RIIβ Arg mutant in a of a in the to in the The and the mutant to the of the the The of the A of of the the A the the A the the and to the to the RIIβ the the mutant a in to in the the and of the binding bound to the A mutant to the the the by of by by the the and the The of the protein and the to the the mutant protein a in the the and the of binding to the A the the A to the A to the the the protein the of the of the protein binding to the of mutant by binding to the A to the of the to to A in the mutant of the the of the RIIβ of the the RIIβ the The of in the mutant the protein of the in mutant the the binding to the of the the the a the of of the conserved in RIIβ the protein and RIIβ of each of the of binding of by binding to the by and by the of the the and the in of the binding to the of the conserved Arg The of the binding and the and mutant a and the and mutant the binding to RIIβ of to binding the and the the in the and of the a of the protein a to the the mutant protein the of RIIβ the the mutant protein a in and a in a in the of the to the of RIIβ the a of a in the to in the The and the mutant to the of the the The of the A of of the the A the the A the the and to the to the RIIβ the the mutant a in to in the the and of the binding bound to the A mutant to the the the by of by by the the and the The of the protein and the to the the mutant protein a in the the and the of binding to the A the the A to the A to the the the protein the of the of the protein binding to the of mutant by binding to the A to the of the to to A in the mutant of the the of the RIIβ The of the the RIIβ the The of in the mutant the protein of the in mutant the the binding to the of the the the a the of cAMP-binding in the of to a in to the of the two binding a each and the two the protein two the of the to the in the the to protein and in the of cAMP-binding A and binding to of conserved to the binding of each and the two in the of the cAMP-binding of the conserved binding the of each and the two The in the the of A in a the and the of the A to a the and RIIβ the of the to a in the cAMP-binding of by a the and B. of the of the of the of of a of by of of a in the of RIIβ to of the in the A the by the in the of the A to RIIβ of the the two and the of the conserved the of the of of to to the A the A the to A the in the the RIIβ to the binding to the A to the A of the mutant protein to the and the of the the and the the of the mutant to the the of A and to of the mutant a in the the and RIIβ The A mutant to the RIIβ bound to the the of the RIIβ bound to the in of the A to binding of the A of the mutant protein the and RIIβ the to the properties the A to the the the of the RIIβ mutant protein and a the binding to the and the of the of the RIIβ to the RIIβ The the in the of the in the the the and of the to the the The and to of to the of and RIIβ the A and in the A and the of the and RIIβ cAMP-binding the of the in the of the and and a each the of the A to the to the by a the of the of the by the each the and a the to of RIIβ the binding to the A in the of a the and the and of the A to the of the binding in RIIβ the A the of the conserved in and RIIβ and Arg in the of the the to the in a in the of the A to the binding the to cAMP-binding A in the to of the two and in a the of the A to the cAMP-binding in in and RIIβ in and and in and in The binding the the the and the A and domains, and the of the the in to to binding and to the in the cAMP-binding to in in the A and in the in RIIβ cAMP-binding the in the to of the of the cAMP-binding domains, of the A binding by cAMP-binding in by the in The and in the A to the of the The conserved to the Arg by in and the in and RIIβ a of the and RIIβ a conserved the and RIIβ a of the two cAMP-binding and of each the in We the a the the and the the the cAMP-binding and in a of and of the of The cAMP-binding in the of to a in to the of the two binding a each and the two the protein two the of the to the in the the to protein and in the of cAMP-binding A and binding to of conserved to the binding of each and the two The in the of the cAMP-binding of the conserved binding the of each and the two The in the the of A in a the and the of the A to a the and RIIβ the of the to a in the cAMP-binding of by a the and B. of the of the of the of of a of by of of a in the of RIIβ to of the in the A the by the The in the of the A to RIIβ of the the two and the of the conserved the of the of of to to the A the A the to A the in the the RIIβ to the binding to the A to the A of the mutant protein to the and the of the the and the the of the mutant to the the of A and to of the mutant a in the the and RIIβ The A mutant to the RIIβ bound to the the of the RIIβ bound to the in of the A to binding of the A of the mutant protein the and RIIβ the to the properties the A to the the the of the RIIβ mutant protein and a the binding to the and the of the of the RIIβ to the RIIβ The the in the of the in the the the and of the to the the The and to of to the of and RIIβ the A and in the A and the of the and RIIβ cAMP-binding the of the in the of the and and a each the of the A to the to the by a the of the of the by the each the and a the to of RIIβ the binding to the A in the of a the and the and of the A to the of the binding in RIIβ A the A the of the conserved in and RIIβ and Arg in the of the the to the in a in the of the A to the binding the to cAMP-binding A in the to of the two and in a the The in to to binding and to the in the cAMP-binding to in in the A and in the in RIIβ cAMP-binding the in the to of the of the cAMP-binding domains, of the A binding by cAMP-binding in by the in The and in the A to the of the The conserved to the Arg by in and the in and RIIβ a of the and RIIβ a conserved the and RIIβ a of the two cAMP-binding and of each the in We the a the the and the the the cAMP-binding and in a of and of the of We and and and in