Dual Specificity A-kinase Anchoring Proteins (AKAPs) Contain an Additional Binding Region That Enhances Targeting of Protein Kinase A 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. 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) subunits. This promotes dissociation of the active catalytic (C) subunits from the tetrameric complex and results in the phosphorylation of substrates in the vicinity of the active kinase (4Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (959) Google Scholar, 5Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). PKA holoenzymes are classified as either type I or type II on the basis of their R subunit composition (RI or RII) (6Skalhegg B.S. Tasken K. Front. Biosci. 2000; 5: D678-D693Crossref PubMed Google Scholar). Four genes encode R subunits (RIα, RIβ, RIIα, and RIIβ). These proteins have distinct physical properties and affinities for cAMP (1Tasken K. Aandahl E.M. Physiol. Rev. 2004; 84: 137-167Crossref PubMed Scopus (628) Google Scholar). Because PKA is a broad specificity serine/threonine protein kinase that regulates a wide range of cellular processes, additional mechanisms have evolved to influence the selectivity of PKA action (7Wong W. Scott J.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (862) Google Scholar). Specificity in PKA action is maintained in part by interaction with protein kinase A anchoring proteins (AKAPs). This family of structurally diverse but functionally related scaffolding proteins targets PKA and other signaling proteins toward distinct substrates. These protein-protein targeting interactions contribute to spatial and temporal regulation of second messenger signaling events (reviewed in Refs. 7Wong W. Scott J.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (862) Google Scholar, 8Jarnaess E. Tasken K. Biochem. Soc. Trans. 2007; 35: 931-937Crossref PubMed Scopus (67) Google Scholar). The AKAP family now includes more than 50 members when including splice variants (7Wong W. Scott J.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (862) Google Scholar, 8Jarnaess E. Tasken K. Biochem. Soc. Trans. 2007; 35: 931-937Crossref PubMed Scopus (67) Google Scholar). Although most of the AKAPs were initially identified on the basis of their ability to bind PKA type II inside cells, it is now recognized that several of these anchoring proteins such as D-AKAP1, D-AKAP2, AKAP220, Ezrin, Merlin, and PAP7 have a dual specificity as they also bind PKA type I (9Gronholm M. Vossebein L. Carlson C.R. Kuja-Panula J. Teesalu T. Alfthan K. Vaheri A. Rauvala Tasken K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. J.A. J. S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, K. J.A. J. S.S. A. 1997; PubMed Scopus Google Scholar, Tasken K. Mol. PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B.S. T. Tasken K. Biol. 2000; PubMed Scopus Google Scholar). AKAPs are to bind RI such as and R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S.S. M.H. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar). of these dual specificity the protein PAP7 and Ezrin Tasken K. Mol. PubMed Scopus Google Scholar, J. M. J. 1997; PubMed Scopus Google Scholar), have been to with PKA type I in R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S.S. M.H. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar). Conventional AKAPs contain a amphipathic helix of that forms the S.M. Scott J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, E.M. Scott J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Scott J.D. Biochem. Soc. Trans. PubMed Scopus Google Scholar). This into a by the R M. Scott J.D. Nat. Biol. PubMed Scopus Google Scholar, M. R. Scott J.D. J. PubMed Scopus Google Scholar). The subunits at the in an an that is for AKAP RI a structurally and E.M. Scott J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Scott J.D. Nat. Biol. PubMed Scopus Google Scholar, M. R. Scott J.D. J. PubMed Scopus Google Scholar, S.S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Tasken K. Carlson C.R. Scott J.D. Mol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). of this to a high affinity and anchoring peptide called were used to an anchoring peptide C.R. T. W. Tasken K. Scott J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). used as to for the PKA type I and PKA type II inside cells. other mechanisms that the amphipathic helix also to binds to the regulatory subunits of PKA a binding of Scott J.D. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), with the PKA holoenzyme J. S.S. M.H. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar). of these AKAPs contain the amphipathic In T cells, PKA type I phosphorylation of in the C-terminal kinase the to T cell T. M. B.S. T. Tasken K. J. PubMed Scopus Google Scholar). This a that of the kinase PKA type I is at in T cells as as in the T. M. B.S. T. Tasken K. J. PubMed Scopus Google Scholar). Activation of the T cells a of the cell and the of PKA type I B.S. Tasken K. T. T. PubMed Scopus Google Scholar). have that the AKAP Ezrin targets PKA type I to the these events A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar). In to binding PKA, Ezrin also with EBP50, a protein that protein associated with glycosphingolipid-enriched Thus, Ezrin, EBP50, and a that the regulatory type in T cell A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar). In this study we a of Ezrin to other AKAPs and an additional the amphipathic helix that RI that such are in a of dual specificity have called this the RI Specifier Region (RISR). In vitro binding show that the RISR is for Ezrin interaction with experiments that in the RISR of Ezrin that RI anchoring the of T cell signaling a type acid of the dual specificity AKAPs Ezrin PAP7 and were the sequence of the used for sequence Mol. Biol. Google Scholar). and a of Cell cells the T were in with bovine and and cells were in from and T cells were by as E.M. B.S. S.S. Tasken K. J. 1998; PubMed Scopus Google Scholar). were from cell T cells were from the cell by with and cells were at with and were and into the of and were into were by cells at were with of were in and with were PKA were in and with the and targeting human Ezrin and a have been A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google and were synthesized Ezrin to were generated by nucleotide and to by in and and and protein were in by and on and as Carlson C.R. Scott J.D. Tasken K. Biochem. J. PubMed Scopus Google Scholar). and of were as Carlson C.R. Scott J.D. Tasken K. Biochem. J. PubMed Scopus Google Scholar, A. T. Vossebein L. Tasken K. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). and Ezrin or protein to were in E. cells, and on used for and assays were synthesized on an and by high were as RISR and were synthesized with on a peptide as R. J. 2002; PubMed Scopus Google Scholar). of interactions and were to and The in the as Carlson C.R. Scott J.D. Tasken K. Biochem. J. PubMed Scopus Google Scholar). with in the of of peptide for on in a of of and of were and on for of were and at for on an were at were by R were as S.M. Scott J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), Scott J.D. Mol. Biol. 1998; Google or bovine to S.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the membrane with peptide or protein in and in Tris-buffered R with catalytic subunit (C) of PKA and in 50 MOPS, 50 and and from by by were at TBS, peptide to the and for the were The were in and the by from cells at with RISR or peptide synthesized in on The were in 50 with and in high PKA R subunit to the were by in and by and of RISR in synthesized in on were with of proteins in binding with were as and binding or and subunit were at a in of with for at of in binding were to the proteins and with for at were with of binding and proteins by in proteins were by and of were in the of at a as T. J. 2004; PubMed Scopus Google Scholar). at and response a of T and T cells were either with of and to the the human T cell and for in or with for A of cells for with or at and with T cell for were and by to the T cells were in and for on cells were on for cells were with in for with for and for with BSA, or in were for were with and in for with fluorescent with a with a at and were and the and of were the for the available were in and a membrane in for by and green green as and the of for green and by the of for green or the of the are as and were by with were and of an in Specificity of Ezrin as the dual specificity AKAP for PKA type I in T cells, were with of the protein A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google to the RI and binding In vitro binding experiments that the Ezrin RI and as by assays a of Ezrin the ability to bind RI Because this the amphipathic helix we that additional in this of the this more a family of by were synthesized on a peptide These were for RI binding to a of in Ezrin that distinct from the amphipathic helix and of and of these the that of at and the interaction with RI of and for RI but more were when of these were experiments that bind to this of Ezrin These results were when a used to show that the acid identified in RI binding by and experiments that the protein with these results that the and the amphipathic helix binding are for RI binding in of the dual specificity AKAPs Ezrin, D-AKAP1, D-AKAP2, and identified a of of the amphipathic helix that anchoring proteins as dual specificity AKAPs contain such a In proteins such as and that the RI in a binding binding by with The in these anchoring proteins were the to produce a sequence Mol. Biol. Google Scholar). The a by the that an acid at a The sequence in the of a of this sequence RI as by the of a sequence to inside cells to this to bind that with the protein in with to a peptide or The of in the and the of in the cell were as and The acid peptide binding the RI Specifier Region as it to enhance binding to RI when with in cells RI and and and of RISR by a peptide A of peptide synthesized in in the peptide by the with the The were for binding of and by of at and RI binding in RI binding in with a in these substitution or were as in to than the sequence in the these in on RI the RISR peptide with to binding of PKA, we a The RISR peptide and the peptide were on and with cell proteins were with and the of PKA subunits by in and subunits were by the RISR peptide but by the peptide In or by the RISR The RISR sequence is in such as and acid M. PubMed Scopus Google Scholar). The of RISR as by in and in the of in the of a acid sequence is that and A. PubMed Scopus Google Scholar, J.L. M. 1990; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). that RISR is of a that augments binding of PKA to the amphipathic of peptide by in a of the PKA Region to were to the binding on RI that with The ability of RISR peptide to a family of proteins in at to the RI anchoring is the amphipathic in C.R. T. W. Tasken K. Scott J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the R subunit and more than as C.R. T. W. Tasken K. Scott J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). binding with a of the R subunit that a of the M. S.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google RISR type and the and proteins with but the more This that RISR with of that are for binding to the amphipathic of this by that the also binds RISR upon RISR binding that RISR with on RI that are C-terminal in the and RISR to as to RI that cAMP binding interaction with the RISR that the RISR and in were by the that RISR peptide in for RI binding to RISR but to interactions with experiments that peptide interaction with RI but on RI binding to that the amphipathic helix binding and RISR have distinct binding in and bind RISR to and R to RISR bind with the amphipathic we the of RISR on R binding in with the amphipathic helix of the and the a of the of RISR with the and amphipathic helix binding that were R binding by of In these the binding of to RI than that of RISR with RIAD, binding by for by of RISR in the RI and and were used as to the that the in RI binding of the in peptide Thus, a on RI affinity by of the RISR and peptide These that RISR and the amphipathic helix RI have the binding properties of the dual specificity an Carlson C.R. Scott J.D. Tasken K. Biochem. J. PubMed Scopus Google Scholar). we the of in this RI binding to the that into the in of for the peptide In the RISR that has RI affinity to the interaction and RI binding to with of and the RISR sequence with either the by or that RISR as an for RI binding and the that the in of in peptide the peptide with also with This to affinity as the from to for as the of of than that of the the of RISR to to the of the RISR to the affinity of Ezrin for RI and we binding with of RI and to type and the RISR and as in that the of the RISR the affinity of Ezrin for RI by the affinity for by this that is a or more in affinity for RI upon of RISR, the for the and interactions into the of the RISR in Ezrin of and with Ezrin forms used to the of the RISR in the subcellular anchoring of These experiments were in of cells were with to the cells of Ezrin A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar). the cells were with mammalian expression Ezrin that is to in the RISR were to Ezrin forms with and in the subcellular of were by the of and the cellular of Ezrin with the Ezrin type and in a that with A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar). when Ezrin in the RISR with a in in the of by of as in and a in by when the RISR in Ezrin Ezrin by in human T cells, by of Ezrin type or Ezrin in the of Ezrin and by for and Although is to in the in T cells, we that a of with Ezrin in the vicinity of the cell membrane that to the PKA type in T cells A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar). with type Ezrin with in the Ezrin to with to the of of PKA and Ezrin to by in T cells when the RISR in Ezrin by substitution of and This the results in experiments and that the RISR have for RI binding to AKAPs in cAMP-mediated of with the RISR events such as This occurs the of a kinase where PKA type I in the kinase to catalytic T. M. B.S. T. Tasken K. J. PubMed Scopus Google Scholar). the family kinase leading to a of T cell Ezrin as the anchoring protein that PKA type I in to by a signaling complex with the protein and the protein A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar). Because PKA type I anchoring to the of this kinase we to of the interaction by the RISR peptide with cAMP-mediated inhibition of T cell function. generated of RISR and the peptide by to the of the T cells were with these for were either with or with of the cAMP of to the were by The peptide inhibition of the peptide experiments were in the of the Ezrin the of Ezrin with the Ezrin with RISR on cAMP-mediated inhibition of T cell the T cells were with or to Ezrin type or cells were with of cAMP as and were with A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar), of Ezrin the of to cAMP and cAMP-mediated inhibition at of cAMP with with at of type Ezrin the to cAMP of Ezrin where the RISR been to and we of Ezrin at the protein with that were and that type and mutant were at the In of PKA type I binding to the RISR in Ezrin either by peptide to the interaction or by of Ezrin with Ezrin the to cAMP-mediated inhibition of T cell that RISR has in In this study we that dual specificity AKAPs contain an additional substitution interaction and we show that the RISR that in Ezrin the of PKA type I in T cells. we that targeting of PKA type I is for the of cAMP-mediated inhibition of T cell function. peptide substitution and we also a RISR that binds RI and to specificity and affinity to RI interaction with RISR to either or a of in the peptide on binding to RI by assays and in Thus, AKAPs that contain have a affinity for RI than AKAPs that contain an amphipathic This additional have evolved to PKA type I to a more than PKA type II These to the helix with the and of RI and and experiments that RI binds AKAP with a affinity and a than the type I holoenzyme complex is S.M. Scott J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Scott J.D. A. PubMed Scopus Google Scholar, A. T. Vossebein L. Tasken K. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). The of an additional binding such as the RISR a for with the RI subunit that the of the PKA type I In the interaction with the amphipathic helix is to the of an PKA holoenzyme complex inside cells (7Wong W. Scott J.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (862) Google Scholar). The of RISR in binding to PKA type I by the that in the Ezrin RISR to binding to RI in vitro and in the amphipathic helix In the Ezrin RISR or binding to and to enhance binding of to the amphipathic PKA binding Ezrin with and and targets PKA type I to this PKA in to for phosphorylation and regulation of A. R. M. Aandahl E.M. Carlson C.R. Tasken K. J. 2007; PubMed Scopus Google Scholar). Thus, it to Ezrin as a to the of RISR in an and in the RISR of Ezrin, PKA type I from this and to cAMP-mediated inhibition of T cell in of by of RISR to cells, a of the type I interaction as when the RISR of Ezrin This of the PKA type I interaction with Ezrin in the complex upon the delivery of RISR the to cAMP-mediated inhibition of T cell In these experiments that the RISR is for PKA type I binding to the dual specificity AKAP to the of the RISR in other AKAPs this the dual specificity AKAP PAP7 has been to bind RI in Tasken K. Mol. PubMed Scopus Google and contain a of has been to RI C.R. T. W. Tasken K. Scott J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and it to the of RISR in this in AKAPs are to functionally related but structurally proteins that a 14-18-amino acid amphipathic S.M. Scott J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Scott J.D. Biochem. Soc. Trans. PubMed Scopus Google Scholar). that the RISR also is of as This is by that the of the in the AKAP PKA binding with that of the by the R subunit that RISR binds to RI a of this of it that RISR binds to RI the by the amphipathic helix as from interaction with RI proteins. RISR that is a of the PKA anchoring Tasken K. Carlson C.R. Scott J.D. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar, S.S. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). it is that the RI with the and by the amphipathic helix This that RISR of the RI in the In of we of and of RISR in the of dual specificity with the of Ezrin and RISR to to the amphipathic helix binding in AKAPs to contain this the RISR C-terminal to the helix also in in a of dual specificity AKAPs such as Ezrin, Merlin, D-AKAP1, and This that this is an that has been into these AKAPs to binding of PKA type I than PKA type II. an AKAP complex PKA type I or type II have as PKA type I at of cAMP than PKA type II. of PKA type I than PKA type II to a AKAP complex the of the physiological by the complex to cAMP. AKAPs with such as and J. S.S. M.H. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar, Scott J.D. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), contain and to have distinct of binding This indicates a in than is to that AKAPs these binding also members of that to contain AKAPs such as AKAP220, and also have or the protein but the of this anchoring and the of the has been B.S. T. Tasken K. Biol. 2000; PubMed Scopus Google Scholar, R. J. M. A. PubMed Scopus Google Scholar, M. K. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, B.S. M. Tasken K. T. J. PubMed Scopus Google Scholar). In show that RISR is an additional in dual specificity and this has for cAMP of by PKA to the dual specificity AKAP are to M. and for for peptide and at the of at of for with R. Carlson for the of this