A Kinase Anchoring Protein (AKAP) Interaction and Dimerization of the RIα and RIβ Regulatory Subunits of Protein Kinase A In vivo by the Yeast Two Hybrid System

Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIα and RIβ as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIα- and RIβ homodimers as well as an RIα:RIβ heterodimer were observed. Single, double and one triple mutation were introduced into the RIα and RIβ subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIα(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIβ were not sufficient to abolish the RIβ dimerization, indicating that additional or other amino acids are important. RIα:RIβ heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIα and RIβ homodimers as well as an RIα:RIβ heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIβ:AKAP149 complex was identified following introduction of RIβ into HEK293 cells. Importantly, RIβ revealed AKAP binding properties similar to those of RIα, indicating that RIβ holoenzymes may be anchored.