Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif (original) (raw)

Abstract

The type I1 CAMP-dependent protein kinase is localized to specific subcellular environments through the binding of the regulatory subunit (RII) dimer to RIIanchoring 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 RIIa. 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 RIIa 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 RIIa binding. These data suggest that anchoring proteins interact with RIIa via an amphipathic helix binding motif. Cyclic AMP serves as the second messenger for numerous hormones and neurotransmitters (1). The CAMP-signaling pathway is composed of individual hormone receptors and adenylate cyclases which are coupled physically through interaction with intermediary GTP-binding proteins (2). Hormone binding to its receptor stimulates adenylate cyclase, resulting in the production of cAMP (2-4). Four molecules of cAMP can bind each dormant PKA'-holoenzyme complex (R,C,), thereby causing release of two active catalytic (C) GM 44427 (to J. D. S.) and EY 03279 (to T. S. A.) and by Medical * This work was supported by National Institutes of Health Grants Research Foundation of Oregon Grant 396-521. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed Vollum Institute

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