A kinase anchoring protein (AKAP) interaction and dimerization of the RIalpha and RIbeta 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 RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(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 RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta:AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored.
Carlson
Cathrine R
CR
Department of Medical Biochemistry, Institute for Basic Medical Sciences, University of Oslo, PO Box 1112 Blindern, N-0317 Oslo, Norway. cathrine.carlson@basalmed.uio.no
Ruppelt
Anja
A
Taskén
Kjetil
K
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection; D020798:Two-Hybrid System Techniques
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D008565:Membrane Proteins; C517834:PRKAR1A protein, human; C517766:PRKAR1B protein, human; D017868:Cyclic AMP-Dependent Protein Kinases