Dual Specificity A-kinase Anchoring Proteins (AKAPs) Contain an Additional Binding Region That Enhances Targeting of Protein Kinase A Type I

摘要

A-kinase anchoring proteins (AKAPs) target protein kinase A (PKA) to a variety of subcellular locations. Conventional AKAPs contain a 14-18-amino acid sequence that forms an amphipathic helix that binds with high affinity to the regulatory (R) subunit of PKA type II. More recently, a group of dual specificity AKAPs has been classified on the basis of their ability to bind the PKA type I and the PKA type II isozymes. In this study we show that dual specificity AKAPs contain an additional PKA binding determinant called the RI Specifier Region (RISR). A variety of protein interaction assays and immunoprecipitation and immunolocalization experiments indicates that the RISR augments RI binding in vitro and inside cells. Cellular delivery of the RISR peptide uncouples RI anchoring to Ezrin leading to release of T cell inhibition by cAMP. Likewise, expression of mutant Ezrin forms where RI binding has been abrogated by substitution of the RISR sequence prevents cAMP-mediated inhibition of T cell function. Thus, we propose that the RISR acts in synergy with the amphipathic helix in dual specificity anchoring proteins to enhance anchoring of PKA type I. A-kinase anchoring proteins (AKAPs) target protein kinase A (PKA) to a variety of subcellular locations. Conventional AKAPs contain a 14-18-amino acid sequence that forms an amphipathic helix that binds with high affinity to the regulatory (R) subunit of PKA type II. More recently, a group of dual specificity AKAPs has been classified on the basis of their ability to bind the PKA type I and the PKA type II isozymes. In this study we show that dual specificity AKAPs contain an additional PKA binding determinant called the RI Specifier Region (RISR). A variety of protein interaction assays and immunoprecipitation and immunolocalization experiments indicates that the RISR augments RI binding in vitro and inside cells. Cellular delivery of the RISR peptide uncouples RI anchoring to Ezrin leading to release of T cell inhibition by cAMP. Likewise, expression of mutant Ezrin forms where RI binding has been abrogated by substitution of the RISR sequence prevents cAMP-mediated inhibition of T cell function. Thus, we propose that the RISR acts in synergy with the amphipathic helix in dual specificity anchoring proteins to enhance anchoring of PKA type I. The second messenger cAMP is frequently utilized in mammalian cells to regulate a variety of physiological processes. Cyclic AMP is generated at the plasma membrane in response to the occupancy of G-protein-coupled receptors. This ultimately leads to the stimulation of adenylyl cyclases, the enzymes that produce cAMP. The newly synthesized cAMP diffuses into the cell where it is available to activate a variety of effector proteins. These include protein kinase A (PKA) 4The abbreviations used are: PKA, protein kinase A; AKAP, A-kinase anchoring protein; RISR, RI specifier region; TFE, trifluoroethanol; RIAD, RI anchoring disrupter; Csk, C-terminal Scr kinase; Lck, lymphocyte-specific protein-tyrosine kinase; EBP50, Ezrin-Radixin-Moesin-binding phosphoprotein 50; siRNA, small interfering RNA; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomains; HEK, human embryonic kidney; GFP, green fluorescent protein; GST, glutathione S-transferase; BSA, bovine serum albumin; TBS, Tris-buffered saline; Ab, antibody; TCR, T cell receptor; MOPS, 4-morpholinepropanesulfonic acid; VSV, vesicular stomatitis virus. (reviewed in Ref. 1Tasken K. Aandahl E.M. Physiol. Rev. 2004; 84: 137-167Crossref PubMed Scopus (628) Google Scholar), cAMP-regulated ion channels (2Kaupp U.B. Seifert R. Physiol. Rev. 2002; 82: 769-824Crossref PubMed Scopus (943) Google Scholar), and Epac guanine nucleotide exchange factors (3de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1634) Google Scholar). Activation of the PKA holoenzyme occurs upon binding of cAMP to the regulatory (R