Activation of protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development in Drosophila. Thus, PKA-anchor protein complexes may be involved in controlling these crucial biological processes. Evaluation of this proposition requires knowledge of PKA binding/targeting proteins in the fly. cDNAs encoding a novel, Drosophila A kinase anchor protein, DAKAP550, have been discovered and characterized. DAKAP550 is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. It binds regulatory subunits (RII) of both mammalian and Drosophila PKAII isoforms. The tethering region of DAKAP550 includes two proximal, but non-contiguous RII-binding sites (B1 and B2). The B1 domain (residues 1406-1425) binds RII ~20-fold more avidly than B2 (amino acids 1350-1369). Affinity-purified anti-DAKAP550 IgGs were exploited to demonstrate that the anchor protein is expressed in many cells in nearly all tissues throughout the lifespan of the fly. However, DAKAP550 is highly enriched and asymmetrically positioned in subpopulations of neurons and in apical portions of cells in gut and trachea. The combination of RII (PKAII) binding activity with differential expression and polarized localization is consistent with a role for DAKAP550 in creating target loci for the reception of signals carried by cAMP. The DAKAP550 gene was mapped to the 4F1.2 region of the X chromosome; flies that carry a deletion for this portion of the X chromosome lack DAKAP550 protein (Han, 1997).
The apparent KD for the DAKAP550…RIIß complex was estimated to be ~10 nM by overlay binding assays. This value is similar to that observed for the binding of RIIß with AKAPs 75 and 79. The size and sequence of the RII-binding domain of DAKAP550 was characterized by a combination of mutagenesis, expression in E. coli, and RII-binding assays. All DAKAP550 cDNAs isolated from bacteriophage expression libraries by functional screening ( i.e., 32P-RIIß overlay binding assays) contain a common 2-kbp fragment. Thus, this segment of DAKAP550 cDNA must encode amino acids that constitute an RII tethering site in the anchor protein. The shared 2-kbp cDNA fragment was excised via digestion with EcoRI and subcloned in pBluescript. Subsequently, the location of the RII tethering site was defined more precisely by applying exonuclease III digestion or polymerase chain reaction methodology to systematically delete 5' and/or 3' portions of the target cDNA. The 2-kbp cDNA fragment and its truncated derivatives were subcloned into the isopropyl-1-thio-ß-D-galactopyranoside-inducible E. coli expression plasmid pET14b. Upstream plasmid DNA encodes a 20-residue peptide that is appended at the N termini of the partial DAKAP550 proteins. The fusion peptide contains six consecutive His residues that enables purification of recombinant, chimeric proteins to near-homogeneity by affinity chromatography on a Ni2+-chelate resin. The shared 2-kbp cDNA encodes a partial protein (amino acids 1001-1676) in DAKAP550 that avidly binds RIIß. This protein was designated fu-B. (Several proteolytic fragments of fu-B also exhibit RIIß binding activity.) Elimination of 161 N-terminal residues and 240 C-terminal residues from fu-B yielded the D1 protein (residues 1162-1436 in DAKAP550). The RIIß binding activities of D1 and fu-B are very similar. However, further deletion of 26 amino acids from the C terminus of D1 generated a protein (D2) whose RII binding activity was diminished by ~90%. Residual RIIß tethering capacity was evident in the D2-3 (residues 1162-1397) and D3 (residues 1162-1381) recombinant proteins. Elimination of all RII binding activity was observed when the C terminus was further truncated to produce the D4 protein (residues 1162-1355). The recombinant, partial DAKAP550 proteins exhibited the same pattern of binding activity when mouse RIIalpha and Drosophila RII were used as the radiolabeled ligands. The tethering region defined by truncation analysis includes two stretches of 20 amino acids (designated B1 and B2), which contain arrangements of large, aliphatic amino acids (Leu, Ile, and Val) that are similar to each other and to patterns observed for previously characterized RII-binding sites. Residues comprising such binding sites are predicted to be assembled as an amphipathic helix by various algorithms. Moreover, mutagenesis/transfection experiments indicate that the creation of a large hydrophobic surface, rather than conservation of a specific primary sequence, governs high-affinity binding of RII isoforms at tethering sites in AKAPs. Computer-based predictions suggest that both the B1 and B2 regions fold into amphipathic helices that contain extended hydrophobic surfaces. However, the two tethering domains do not contribute equally to overall RII binding activity. A partial DAKAP550 protein that contains B1, but lacks B2, complexes 32P-RII subunits nearly as avidly as a polypeptide (D1) that includes both B1 and B2. This result and the 90%-95% decrease in RII binding activity associated with selective disruption of B1 in the D2 protein) demonstrate that amino acids 1406-1425 constitute the high-affinity tethering site in the Drosophila anchor protein (Han, 1997).
Thus, the principal RII tethering domain maps to a region of DAKAP550 corresponding to amino acid residues 1406-1425 (B1). The arrangement of amino acids with long, aliphatic side chains (Leu, Ile, and Val) in B1 matches the distribution of critical, Leu, Ile, and Val residues in the RII tethering sites of AKAP75 and other mammalian AKAPs. Substitution of any of these residues with Ala eliminates or sharply reduces the ability of AKAP75 to sequester RII subunits. When the integrity of the B1 tethering site in DAKAP550 is disrupted by mutation, RII binding affinity declines by at least an order of magnitude. In the absence of B1, a reduced, but detectable level of RII binding avidity is contributed by a proximal RII-binding site (residues 1350-1369) designated B2. In contrast, partial DAKAP polypeptides containing either B1 alone or both B1 and B2 bind similar amounts of the RIIß ligand. Thus, under standard assay conditions the B1 tethering region plays a predominant role in sequestering RII subunits. The precise function of B2 remains to be determined. One speculative possibility is that in intact cells (where the PKAII concentration is higher than that used in overlay binding assays) B2 may provide a site for transient, rapidly reversible accumulation of RII subunits (Han, 1997).
Secondary structure predictions suggest that residues 1350-1369 (B2) and 1406-1425 (B1) in DAKAP550 fold as amphipathic helices with large hydrophobic surfaces. Mutagenesis and binding studies on mammalian AKAPs 75 and 79 indicate that both a large hydrophobic surface at the tethering site and a complementary hydrophobic surface on RIIß and RIIalpha dimers are essential for formation of stable AKAP-PKAII complexes. Evidently, binding of DAKAP550 with Drosophila RII (and mammalian RII isoforms) is driven by similar hydrophobic interactions. The preferential binding of RIIß with B1 (versus B2) indicates that the 'core' ligand affinity contributed by the hydrophobic surface is further modulated by additional features in the structure of DAKAP550. Possible factors involved in modulating affinities of B1 and B2 for RII are: (1) substitution of conserved Val or Leu with Thr at the C terminus of B2; (2) the presence of 10 hydrophobic residues on the surface of the predicted B1 amphipathic helix, as compared with 8 hydrophobic amino acids at the corresponding region of B2; (3) differences in non-conserved amino acids in the 20-residue tethering domains, and (4) stabilization/destabilization of RII-tethering site interactions by flanking segments of the DAKAP550 polypeptide. Further systematic studies, employing mutagenesis, protein expression, and RII binding analyses, will be required to identify amino acid residues that enhance or diminish the affinity of the B1 and B2 domains for RII isoforms (Han, 1997).
The RII-binding domain is inserted between two lengthy blocks of DAKAP550 sequence (residues 892-955 and 1693-1929) that are homologous with portions of both C. elegans F10F2.1 and the human beige-like protein (BLP). F10F2.1 lacks a comparable tethering site for RII subunits. However, this is expected because C. elegans has a single R subunit gene, which encodes a cAMP-binding protein that is a homolog of mammalian RI, not RII. The inclusion of DAKAP550 homology regions in BLP raises the possibility that the previously uncharacterized human protein may (by analogy) be involved in cAMP-mediated signaling. BLP contains a partially conserved, candidate RII binding sequence that should be amenable to characterization by mutagenesis and overlay binding assays (Han, 1997).
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