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Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding1

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Abstract

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIα very well (apparent KD values of 0.5 and 2 nM, respectively). Both proteins also bind RIIβ quite well, but with three- to fourfold lower affinities than those observed versus RIIα. However, only S-AKAP84/D-AKAP1 interacts with RIα at a nanomolar affinity (apparent KD of 185 nM). In comparison, AKAP95 binds RIIα (apparent KD of 5.9 nM) with a tenfold higher affinity than RIIβ and has no detectable binding to RIα. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIα and RIIβ to all the AKAPs examined (EC50-values from 6 to 360 nM). Furthermore, RIα interaction with S-AKAP84/D-AKAP1 was competed (EC50 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.

Introduction

Cyclic AMP regulates a number of discrete physiological responses through cAMP-dependent protein kinase (PKA). Although PKA has broad substrate specificity, one intriguing aspect of its action is the ability to phosphorylate selectively individual substrates in response to distinct hormonal stimuli. Selectivity in PKA action may be mediated by particular pools of kinase compartmentalized at different subcellular loci through interaction with A-kinase anchoring proteins (AKAPs, reviewed in Colledge and Scott 1999, Rubin 1994) which target PKA towards specific substrates. The PKA holoenzyme complex forms a tetramer consisting of two regulatory (R) and two catalytic (C) subunits. Four different regulatory subunits (RIα, RIβ, RIIα and RIIβ) of PKA have been identified and serve to regulate catalytic activity by binding and inactivating the C-subunit. The C-subunit is released and activated upon the binding of four molecules of cAMP to the R-subunit dimer (for review and references, see Francis and Corbin 1994, Scott 1991, Taylor et al 1992).

Targeting of PKA to various subcellular loci is mediated by interaction of the R-subunits with different AKAPs Colledge and Scott 1999, Rubin 1994. Whereas PKA type I (containing RIα or RIβ) is known to be mainly soluble, it has also been demonstrated to localize in proximity to membrane receptors such as antigen receptors on lymphoid cells and nicotinic acetylcholine receptors in neuromuscular junctions Skalhegg et al 1994, Imaizumi-Scherrer et al 1996. In contrast, PKA type II (containing RIIα or RIIβ) is primarily particulate and associated with cytoskeletal elements and a number of organelles. While the subcellular targeting of RI via mono or dual-specific AKAPs is emerging Angelo and Rubin 1998, Huang et al 1997a, Huang et al 1997b, Miki and Eddy 1998, Miki and Eddy 1999, it is well known that RII can be specifically bound to AKAPs that bind exclusively RII or both RII and RI. AKAPs that bind RII have been localized to microtubules (MAP2 and AKAP150), postsynaptic densities and cortical actin (AKAP79/75), actin (AKAP78/ezrin, AKAP-KL), nuclear matrix (AKAP95 and nuclear AKAP150), sarcoplasmic reticulum and nuclear envelope (mAKAP), endoplasmic reticulum (D-AKAP1), peroxisomes (AKAP220), Golgi apparatus (AKAP85), mitochondria (S-AKAP84/149/D-AKAP1), centrosomes (AKAP450), filopodia (gravin/AKAP250), sperm flagella (AKAP110, FSC1/AKAP82) and shown to target to membrane receptors as β2-adrenoreceptors (gravin/AKAP250), and various ion channels (AKAP15/18, AKAP79, yotiao/AKAP450, ezrin) (for review and references, see Colledge and Scott 1999, Fraser and Scott 1999).

The interaction of PKA RII subunits with various AKAPs is shown to involve an amphipathic helix motif with a conserved structure (X{L,I,V}X3 {A,S}X2{L,I,V}{L,I,V}X2{L,I,V}{L,I,V}X2{A,S}{L,I,V}) in the AKAP where the hydrophobic face binds the RII dimer Carr et al 1991, Vijayaraghavan et al 1999 Furthermore, the recent solution of the structure of the dimerization and AKAP binding domains of RIIα have shown that high-affinity AKAP binding involves an X-type four-helix bundle dimerization motif with an extended hydrophobic face in residues 1-44 (Newlon et al., 1999). In contrast to the interaction of RIIα with AKAPs, the RIα N terminus has a stable, α-helical dimerization and docking motif that involves residues 12 to 61 and includes two stable disulfide bonds (Leon et al., 1997). In addition to the distinct subcellular distribution of PKA type I and II, isozymes with RIIα and RIIβ have also been demonstrated to localize differently in the Golgi-centrosomal area (Keryer et al., 1999). A recent study addressed the specificity of the interaction of the dimerization/AKAP binding domain of RIIα and RIIβ and showed clearly that, whereas some anchoring proteins bind both RIIα and RIIβ, although with different affinity, other anchoring molecules such as AKAP95 almost exclusively bind RIIα (Hausken et al., 1996). The biochemical basis for the distinct distribution of PKA isozymes is based on the specificity and affinity in PKA interaction with available AKAPs. In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes, we found it of importance to develop methods to assess the apparent binding constants of the R-AKAP interaction. We present a novel approach to examine directly binding of AKAPs to all R-subunit isoforms with a high degree of accuracy where the R-subunit is immobilized on a modified cAMP surface, and the association and dissociation of AKAP proteins is examined by surface plasmon resonance. We examine the PKA-interaction of AKAP79, reported to bind both RIIα and RIIβ, with the four R-subunits (RIα, RIβ, RIIα and RIIβ) and compare it with that of AKAP95, reported to bind more selectively RIIα, and with that of S-AKAP84/D-AKAP1 (AKAP121, AKAP149), which is reported as a dual-specific AKAP binding both RI and RII (Huang et al., 1997b) and show selectivity in AKAP binding for the different R-subunits. Apparent binding constants and EC50 values for competitor peptides are determined.

Section snippets

Results

The number of A-kinase anchoring proteins (AKAPs) is growing fast and several of these highly specific PKA-binding proteins are in the same cellular compartment, sharing the four available R-subunits, RIα, RIβ, RIIα and RIIβ. Therefore accurate numbers for AKAP/R-subunit interactions are needed to understand the basis for differential subcellular distribution of PKA R-subunits. We expressed fragments of S-AKAP84/D-AKAP1, AKAP79 and AKAP95 containing the respective RII binding domains as

Discussion

Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. The formation of these complexes is mediated by specialized protein motifs that participate in protein-protein interactions. The N-terminal regions of both types of R-subunit, the type I and II, bind to amphipathic α-helical motifs in different AKAP proteins. However, distinct distributions of PKA isozymes are observed based on differences in

Reagents

8-AHA-cAMP (8-aminohexyl amino adenosine 3′-,5′-cyclic monophosphate) was purchased from Biolog, Bremen, Germany, the peptide substrate, LRRASLG, from Bachem Biochemicals, NHS, EDC and Surfactant P20, Sensor chips CM 5 research grade from Biacore AB, Sweden. Other reagents were purchased as follows: ATP and cAMP (Sigma), PMSF (Boehringer Mannheim), media supplies (Difco). All other reagents were obtained in the purest grade available.

Expression and purification of A-kinase anchoring proteins

Human cDNA clones encoding AKAP79 and S-AKAP84/D-AKAP1 (K.

Acknowledgements

Attila Toth and Claudia Hahnefeld for excellent assistance on SPR, Bastian Zimmermann for helpful discussions and Susan S. Taylor for kindly providing expression vectors for RIα, RIIα and RIIβ. This work was supported by grants SFB 394 B4 from the Deutsche Forschungsgemeinschaft and the FoRUM (to F.W.H.) and by grants from the Norwegian Research Council, Norwegian Cancer Society, Anders Jahre’s Foundation and Novo Nordisk Research Foundation (to K.T.).

References (43)

  • K.L. Guan et al.

    Eukaryotic proteins expressed in Escherichia colian improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

    Anal. Biochem.

    (1991)
  • Z.E. Hausken et al.

    Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII - classification of side chain determinants for anchoring and isoform selective association with AKAPs

    J. Biol. Chem.

    (1996)
  • L.J. Huang et al.

    Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits

    J. Biol. Chem.

    (1997)
  • R. Karlsson et al.

    Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors

    J. Immunol. Methods

    (1997)
  • G. Keryer et al.

    Differential localization of protein kinase A type II isozymes in the Golgi-centrosomal area

    Exp. Cell. Res.

    (1999)
  • G. Keryer et al.

    Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase

    J. Biol. Chem.

    (1998)
  • D.A. Leon et al.

    A stable alpha-helical domain at the N terminus of the RIalpha subunits of cAMP-dependent protein kinase is a novel dimerization/docking motif

    J. Biol. Chem.

    (1997)
  • K. Miki et al.

    Identification of tethering domains for protein kinase A type Ialpha regulatory subunits on sperm fibrous sheath protein FSC1

    J. Biol. Chem.

    (1998)
  • K. Miki et al.

    Single amino acids determine specificity of binding of protein kinase A regulatory subunits by protein kinase A anchoring proteins

    J. Biol. Chem.

    (1999)
  • M.G. Newlon et al.

    The A-kinase anchoring domain of type IIalpha cAMP-dependent protein kinase is highly helical

    J. Biol. Chem.

    (1997)
  • L.D. Saraswat et al.

    Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

    J. Biol. Chem.

    (1986)
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