Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif.
The type II cAMP-dependent protein kinase is localized to specific subcellular environments through the binding of the regulatory subunit (RII) dimer to RII-anchoring proteins. Computer-aided analysis of secondary structure, performed on four RII-anchoring protein sequences (the microtubule-associated protein 2, P150, and two thyroid proteins Ht 21 and Ht 31), has identified common regions of approximately 14 residues which display high probabilities of forming amphipathic helices. The potential amphipathic helix region of Ht 31 (Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile) lies between residues 494 and 507. A bacterially expressed 318-amino acid fragment, Ht 31 (418-736), containing the amphipathic helix region, was able to bind RII alpha. Site-directed mutagenesis designed to disrupt the secondary structure in the putative binding helix reduced binding dramatically. Specifically, substitution of proline for Ala-498 significantly diminished RII alpha binding, and similar mutation of Ile-502 or Ile-507 abolished interaction. Mutation of Ala-522 to proline, which is located outside the predicted amphipathic helix region, had no effect on RII alpha binding. These data suggest that anchoring proteins interact with RII alpha via an amphipathic helix binding motif.
Carr
D W
DW
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201-3098.
Stofko-Hahn
R E
RE
Fraser
I D
ID
Bishop
S M
SM
Acott
T S
TS
Brennan
R G
RG
Scott
J D
JD
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases