Molecular Characterization of a Novel A Kinase Anchor Protein from Drosophila melanogaster
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26611
ISSN1083-351X
AutoresJing-Dong J. Han, Nicholas E. Baker, Charles S. Rubin,
Tópico(s)Developmental Biology and Gene Regulation
ResumoActivation of protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development inDrosophila. 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. We now report the discovery and characterization of cDNAs encoding a novel, DrosophilaA kinase anchor protein, DAKAP550. DAKAP550 is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. It binds regulatory subunits (RII) of both mammalian andDrosophila 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. Activation of protein kinase A (PKA) at discrete intracellular sites facilitates oogenesis and development inDrosophila. 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. We now report the discovery and characterization of cDNAs encoding a novel, DrosophilaA kinase anchor protein, DAKAP550. DAKAP550 is a large (>2300 amino acids) acidic protein that is maximally expressed in anterior tissues. It binds regulatory subunits (RII) of both mammalian andDrosophila 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. Cyclic AMP-dependent protein kinase (PKA) 1The abbreviations used are: PKA, cAMP-dependent protein kinase; R, regulatory subunit of cAMP-dependent protein kinase; C, catalytic subunit of cAMP-dependent protein kinase; AKAP, A kinase anchor protein; DAKAP550, Drosophila A kinase anchor protein with an apparent M r of 550,000; bp, base pair(s); kbp, kilobase pair(s); GST, glutathione S-transferase; BLP, human beige-like protein. 1The abbreviations used are: PKA, cAMP-dependent protein kinase; R, regulatory subunit of cAMP-dependent protein kinase; C, catalytic subunit of cAMP-dependent protein kinase; AKAP, A kinase anchor protein; DAKAP550, Drosophila A kinase anchor protein with an apparent M r of 550,000; bp, base pair(s); kbp, kilobase pair(s); GST, glutathione S-transferase; BLP, human beige-like protein. is the principal mediator of actions of hormones and neurotransmitters that activate adenylate cyclase (1Beebe S.J. Corbin J.D. Boyer P.D. The Enzymes. 17. Academic Press, Orlando, FL1986: 44-100Google Scholar, 2Edelman A.M. Blumenthal D.K. Krebs E.G. Annu. Rev. Biochem. 1987; 56: 567-613Crossref PubMed Scopus (1017) Google Scholar, 3Taylor S.S. Buechler J.A. Yonemoto Y. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (956) Google Scholar, 4Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (408) Google Scholar). Signals borne by cAMP are often targeted to effectors that accumulate at discrete intracellular locations (5Rubin C.S. Biochim. Biophy. Acta. 1994; 1224: 467-479PubMed Google Scholar, 6Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar, 7Faux M. Scott J.D. Cell. 1996; 85: 9-12Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In part, targeting is accomplished by establishing a non-uniform distribution of PKA molecules within cells. Such asymmetry is evident in cells where PKAIIα and PKAIIβ isoforms 2PKA isoforms are named according to their homodimeric R subunits. Distinct genes encode the RIIα, RIIβ, RIα, and RIβ proteins. 2PKA isoforms are named according to their homodimeric R subunits. Distinct genes encode the RIIα, RIIβ, RIα, and RIβ proteins. are attached to cytoskeleton and/or organelles via A kinase anchor proteins (AKAPs) (5Rubin C.S. Biochim. Biophy. Acta. 1994; 1224: 467-479PubMed Google Scholar,6Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar). Prototypic neuronal anchor proteins (AKAPs 75, 79, and 150) possess a binding site for regulatory subunits (RII) of PKAII isoforms and distinct domains that non-covalently link AKAP·PKAII complexes to the microtubule-based dendritic cytoskeleton of neurons and the cortical actin cytoskeleton of non-neuronal cells (5Rubin C.S. Biochim. Biophy. Acta. 1994; 1224: 467-479PubMed Google Scholar, 6Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar, 7Faux M. Scott J.D. Cell. 1996; 85: 9-12Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Bregman D.B. Battacharya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Abstract Full Text PDF PubMed Google Scholar, 9Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Abstract Full Text PDF PubMed Google Scholar, 10Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 11Glantz S.B. Li Y. Rubin C.S. J. Biol. Chem. 1993; 268: 12796-12804Abstract Full Text PDF PubMed Google Scholar, 12Glantz S.B. Amat J.A. Rubin C.S. Mol. Biol. Cell. 1992; 3: 1215-1228Crossref PubMed Scopus (108) Google Scholar, 13Li Y. Ndubuka C. Rubin C.S. J. Biol. Chem. 1996; 271: 16862-16869Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Both cytoskeletal locations are closely apposed to the plasma membrane. Thus, anchored PKAII is placed in proximity with a signal generator (adenylate cyclase) and multiple PKA substrate/effector proteins (e.g.myosin light chain kinase, microtubule-associated protein-2, ion channels, serpentine receptors that couple with Gs and adenylate cyclase). This arrangement generates target sites for cAMP action (5Rubin C.S. Biochim. Biophy. Acta. 1994; 1224: 467-479PubMed Google Scholar, 6Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar, 14Rosenmund C. Carr D.W. Bergeson L.E. Nilaver G. Scott J.D. Westbrook G.L. Nature. 1994; 368: 853-856Crossref PubMed Scopus (323) Google Scholar). A distinct group of RII-binding proteins mediates association of PKAII isoforms with mitochondria, Golgi membranes, peroxisomes, centrioles, and other organelles in numerous cell types (5Rubin C.S. Biochim. Biophy. Acta. 1994; 1224: 467-479PubMed Google Scholar, 6Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar, 15Lin R.-Y. Moss S.B. Rubin C.S. J. Biol. Chem. 1995; 270: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 16Lester L.B. Coghlan V.M. Nauert B. Scott J.D. J. Biol. Chem. 1996; 271: 9460-9465Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Thus, anchored PKAs appear to be involved in the regulation multiple cell functions. Although substantial progress has been made in identifying and characterizing AKAPs, knowledge of anchor protein functions in vivo is limited. One approach toward linking AKAP structure with physiological function is to complement studies on PKA anchoring in mammalian systems with investigations on a lower organism that is amenable to concerted biochemical, cellular, molecular, and genetic analysis. Drosophila melanogaster provides an attractive model system because classical and molecular genetics have implicated PKA in signaling pathways that (a) generate cell or tissue polarity and/or (b) require accumulation of PKA at a discrete intracellular location (17Perrimon N. Cell. 1995; 80: 517-520Abstract Full Text PDF PubMed Scopus (144) Google Scholar, 18Li W. Ohlmeyer J.T. Lane M.E. Kalderon D. Cell. 1995; 80: 553-562Abstract Full Text PDF PubMed Scopus (279) Google Scholar, 19Lane M.E. Kalderon D. Genes Dev. 1994; 8: 2986-2995Crossref PubMed Scopus (117) Google Scholar, 20Lane M.E. Kalderon D. Mech. Dev. 1995; 49: 191-200Crossref PubMed Scopus (36) Google Scholar, 21Perrimon N. Cell. 1996; 86: 513-516Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). For example, localized activation of PKA negatively modulates hedgehog signaling during Drosophila development. Hedgehog is a secreted protein that binds with serpentine receptors in plasma membrane. Receptor occupancy activates a signaling pathway that elicits expression of genes (dpp, wg etc.) that control anterior-posterior patterning in developing tissues. PKA accumulates selectively along segments of plasma membrane (or underlying cytoskeleton) that constitute adhesive junctions between oocytes and supporting nurse cells (20Lane M.E. Kalderon D. Mech. Dev. 1995; 49: 191-200Crossref PubMed Scopus (36) Google Scholar). Localized PKA activity appears to be essential for assembly and stabilization of intercellular bridges at these junctions. The bridges enable the flow of critical proteins and RNAs from nurse cells to developing oocytes. Finally, PKA-mediated phosphorylation controls a re-orientation of microtubule organizing centers in germ cells during oogenesis (19Lane M.E. Kalderon D. Genes Dev. 1994; 8: 2986-2995Crossref PubMed Scopus (117) Google Scholar). Remodeling of the microtubule-based cytoskeleton generates a polarized structure that enables differential segregation of specific mRNAs to anterior or posterior locations via kinesin motors. Subsequent translation of spatially-segregated mRNAs establishes gradients of proteins (e.g. biocoid, oskar) that are essential for normal morphogenesis and development. It has been suggested that a subset of PKA molecules, that is anchored at the microtubule organizing center, governs the polarity and positioning of microtubules via the phosphorylation of microtubule-associated proteins (19Lane M.E. Kalderon D. Genes Dev. 1994; 8: 2986-2995Crossref PubMed Scopus (117) Google Scholar). The cited studies suggest that PKA-anchor protein complexes may be intimately involved in controlling critical aspects ofDrosophila reproduction and development. No information is currently available concerning RII (PKAII)-binding proteins in the fly. Thus, initial steps in evaluation of the PKA anchoring model entail the identification and basic characterization of DrosophilaAKAPs (DAKAPs). We now report the discovery of a novelDrosophila AKAP (DAKAP550), the cloning and sequencing of DAKAP550 cDNA, the differential expression of DAKAP550 protein during embryogenesis, the chromosomal location of the DAKAP550 gene, and the mapping of a binding site in DAKAP550 that complexes both mammalian and Drosophila RII subunits. Expression libraries of D. melanogaster (Canton S strain) cDNAs were searched for inserts encoding A kinase anchor proteins by both functional (RII binding) assays and classical DNA hybridization. cDNAs in the libraries were generated by reverse transcription of template mRNAs isolated from embryos 0–24 h after fertilization. Initially, cDNA libraries in bacteriophages λZAP (provided by Dr. R. Reinke, Deptartment of Developmental Molecular Biology, Albert Einstein College of Medicine, Bronx, NY) and λgt11 (CLONTECH) were screened by the procedure of Bregman et al. (8Bregman D.B. Battacharya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Abstract Full Text PDF PubMed Google Scholar, 9Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Abstract Full Text PDF PubMed Google Scholar). This method detects AKAP-β-galactosidase fusion proteins in phage plaques by their ability to bind 32P-labeled RIIβ. Six recombinant phage clones (see Fig. 2, below) that contain overlapping cDNA sequences were retrieved from an initial population of 106 plaques and were characterized. A 5′ EcoRI-NcoI fragment (300 bp) from clone Z cDNA (Fig. 2) was employed as a template to synthesize a random-primed 32P-labeled probe for further screening. Screening via DNA hybridization (22Lu X. Gross R.E. Bagchi S. Rubin C.S. J. Biol. Chem. 1990; 265: 3293-3303Abstract Full Text PDF PubMed Google Scholar) yielded 13 independent cDNA clones from a 5′ stretched cDNA library in bacteriophage λgt11 (CLONTECH). Four of these cDNAs were characterized as described under “Results.” A filter, which contains a gridded array of DNA fragments (in cosmids) that span the Drosophila genome (23Hoheisel J.D. Lennon G.G. Zehetner G. Lehrach H. J. Mol. Biol. 1991; 220: 903-914Crossref PubMed Scopus (58) Google Scholar), was provided by Dr. J. Hoheisel (University of Heidelberg, Heidelberg, Germany). Two overlapping genomic DNA clones (named 64G6 and 81C9) hybridized strongly with each of three 32P-labeled cDNA probes: the cDNA insert from clone II-1 and the 2- and 1-kbp cDNA fragments released from clone number 8 by EcoRI digestion (see Fig. 2, below). Collectively, these cDNAs encode a segment of DAKAP550 that includes >2000 amino acids. Cosmids 64G6 and 81C9 were obtained from Dr. J. Hoheisel. Complementary DNA inserts from recombinant λZAP phage were isolated in the plasmid pBluescript SK (Stratagene) via M13 phage-promoted, plasmid excision inEscherichia coli (24Short J.M. Sorge J.A. Methods Enzymol. 1992; 216: 495-508Crossref PubMed Scopus (63) Google Scholar). cDNA inserts obtained from λgt11 phage clones were subcloned into the plasmid pGEM7Z (Promega). Nested deletions were prepared by exonuclease III digestion according to the procedure of Henikoff (25Henikoff S. Methods Enzymol. 1987; 155: 156-165Crossref PubMed Scopus (676) Google Scholar). Intact and truncated cDNAs and genomic DNA fragments were sequenced by a dideoxynucleotide chain termination procedure (26Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52610) Google Scholar) using T7, T3, SP6, and custom oligonucleotide primers. Taq Dye Deoxy Terminator Cycle Sequencing Kits (Applied Biosystems) were used according to the manufacturer's instructions. DNA products were separated and analyzed in a model 377 automated DNA Sequencer (Applied Biosystems) in the DNA Analysis Facility of Albert Einstein College of Medicine. Analysis of sequence data, sequence comparisons, and data base searches were performed using PCGENE-IntelliGenetics software (IntelliGenetics, Mountainview, CA) and BLAST programs (27Altschul S.R. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70322) Google Scholar) provided by the NCBI server (National Institutes of Health). Overlay binding assays have been described in several papers (8Bregman D.B. Battacharya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Abstract Full Text PDF PubMed Google Scholar, 9Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Abstract Full Text PDF PubMed Google Scholar). In brief, a Western blot is probed with 32P-labeled RIIβ (using a subunit concentration of 0.3 nm and 2 × 105 cpm of 32P-radioactivity/ml) and RIIβ-binding proteins are visualized by autoradiography. Results were quantified by scanning laser densitometry (Pharmacia LKB Ultroscan XL laser densitometer) or PhosphorImager analysis (Molecular Dynamics) as described previously (28Ndubuka C. Li Y. Rubin C.S. J. Biol. Chem. 1993; 268: 7621-7624Abstract Full Text PDF PubMed Google Scholar). Polytene chromosomes were isolated from salivary glands of second instar Drosophila larvae as described by Ashburner (29Ashburner M. Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 44-49Google Scholar). DAKAP550 cDNAs were labeled with biotinyl-16-dUTP (Boehringer Mannheim) via DNA polymerase I-catalyzed nick translation. Chromosomes were hybridized with the biotinylated cDNAs indicated under “Results” for 16 h at 58 °C, using conditions described by Ashburner (29Ashburner M. Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 44-49Google Scholar). Gene-cDNA complexes were visualized by using a Detek I-horseradish peroxidase kit (Enzo Biochemicals) according to the manufacturer's instructions. The basis for detection of the gene is that biotinylated DAKAP550 cDNA hybridized with complementary sequences in DAKAP550 exons will avidly bind streptavidin-conjugated peroxidase. Subsequent incubation with diaminobenzidine and H2O2 results in peroxidase-catalyzed synthesis of an insoluble precipitate at the site of cDNA-genomic DNA hybridization. Photographs of hybridized, peroxidase-stained chromosomes were obtained with a Zeiss axioplan microscope, using phase-contrast optics. The peroxidase-generated precipitate appears as an intense black stripe on the chromosome (see Fig. 4, below). A cDNA fragment encoding amino acids 1472–1676 in DAKAP550 (see Fig. 3 below) was excised from clone number 8 (Fig. 2) by digestion with BamHI and EcoRI and was subcloned into the expression plasmid pGEX2T (Pharmacia), which was cleaved with the same enzymes. This places cDNA encoding the partial DAKAP550 polypeptide downstream from and in-frame with the 3′ terminus of a glutathione S-transferase (GST) gene in the vector. Transcription of the GST-fusion gene is driven by an inducibletac promoter. A high level of chimeric GST-partial DAKAP550 polypeptide (designated fu-A) was produced when E. coliDH5α was transformed with recombinant pGEX2T plasmid and then induced with 0.5 mmisopropyl-1-thio-β-d-glactopyranoside as described previously (30Land M. Islas-Trejo A. Freedman J.H. Rubin C.S. J. Biol. Chem. 1994; 269: 9234-9244Abstract Full Text PDF PubMed Google Scholar). After induction, bacteria were disrupted in a French press and the soluble fu-A protein (∼5 mg) was purified to homogeneity by affinity chromatography on glutathione-Sepharose 4B beads (Pharmacia) (30Land M. Islas-Trejo A. Freedman J.H. Rubin C.S. J. Biol. Chem. 1994; 269: 9234-9244Abstract Full Text PDF PubMed Google Scholar). A cDNA fragment encoding residues 1001–1676 in DAKAP550 (Fig. 3) was cloned into the expression plasmid pET14b as described previously (15Lin R.-Y. Moss S.B. Rubin C.S. J. Biol. Chem. 1995; 270: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 31Li Y. Rubin C.S. J. Biol. Chem. 1995; 270: 1935-1944Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This enables isopropyl-1-thio-β-d-galactopyranoside-induced synthesis of a (His)6-tagged partial DAKAP550 fusion protein (designated fu-B) in E. coli BL21 (DE3) transformed with recombinant pET14b. The fusion protein is described in more detail under “Results.” Induced E. coli BL21 were harvested, disrupted, and separated into soluble and particulate fractions (30Land M. Islas-Trejo A. Freedman J.H. Rubin C.S. J. Biol. Chem. 1994; 269: 9234-9244Abstract Full Text PDF PubMed Google Scholar). fu-B protein was recovered in the soluble fraction and purified to near-homogeneity by nickel-chelate chromatography as previously reported (31Li Y. Rubin C.S. J. Biol. Chem. 1995; 270: 1935-1944Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Several batches of purified fu-B (∼6 mg of protein from 1 liter of E. coli) were coupled to CNBr-activated Sepharose 4B as described previously (9Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Abstract Full Text PDF PubMed Google Scholar). Purified fu-A protein (see above) was injected into rabbits (0.35 mg of initial injection; 0.2 mg for each of three booster injections) at Covance Laboratories (Vienna, VA) to generate antisera. Serum was collected at 3-week intervals. Antibodies that bind GST were eliminated by passing serum over a column of GST-Sepharose 4B (30Land M. Islas-Trejo A. Freedman J.H. Rubin C.S. J. Biol. Chem. 1994; 269: 9234-9244Abstract Full Text PDF PubMed Google Scholar). Next, adsorbed serum was applied to a column of fu-B Sepharose 4B (see above). The fu-B protein includes the complete sequence of fu-A (and therefore, all fu-A epitopes). The resin was extensively washed and anti-DAKAP550 IgGs were then isolated by successive elutions at pH 2.5 and 11.8, as described previously (30Land M. Islas-Trejo A. Freedman J.H. Rubin C.S. J. Biol. Chem. 1994; 269: 9234-9244Abstract Full Text PDF PubMed Google Scholar). Deletion mutagenesis was performed via exonuclease III digestion or polymerase chain reaction as described previously (11Glantz S.B. Li Y. Rubin C.S. J. Biol. Chem. 1993; 268: 12796-12804Abstract Full Text PDF PubMed Google Scholar,31Li Y. Rubin C.S. J. Biol. Chem. 1995; 270: 1935-1944Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Mutants were verified by DNA sequencing. Amplified cDNAs were cloned in pET14b and fusion proteins were expressed in E. coli and purified, as described previously (31Li Y. Rubin C.S. J. Biol. Chem. 1995; 270: 1935-1944Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Intact Drosophila or separated fly heads and bodies were suspended in 4 volumes of buffer A (20 mmsodium phosphate, pH 7.4, 20 mm NaCl, 0.2 mmdithiothreitol, 1 mm EDTA, 0.2 mm EGTA, 10 μg/ml soybean trypsin inhibitor, 40 μg/ml aprotinin, 10 μg/ml pepstatin A, and 40 μg/ml leupeptin) and disrupted in a Polytron homogenizer (two 30-s cycles of homogenization at the maximum setting). All operations were performed at 0–4 °C. The homogenate was centrifuged at 12,000 × g for 20 min and the supernatant solution (cytosol) was collected. The pellet was resuspended in the original volume of buffer A and then was homogenized and centrifuged as described above. The resulting supernatant solution (designated “wash”) was collected; the pelleted, particulate fraction of Drosophila homogenates was dispersed in the starting volume of buffer A by a final round of homogenization. The concentration of protein in subcellular fractions fromDrosophila and in purified samples of partial DAKAP550 polypeptides (see above) was determined by the method of Bradford (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215608) Google Scholar). Proteins were denatured in gel loading buffer and subjected to electrophoresis in 5.5, 10, or 12% polyacrylamide gels containing 0.1% SDS as described previously (8Bregman D.B. Battacharya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Abstract Full Text PDF PubMed Google Scholar,9Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Abstract Full Text PDF PubMed Google Scholar). Phosphorylase b (M r = 97,000), transferrin (77,000), albumin (68,000), ovalbumin (45,000), carbonic anhydrase (29,000), and cross-linked phosphorylase (195,000–584,000) were used as standards for the estimation of M rvalues. Size-fractionated proteins were transferred from denaturing polyacrylamide gels to an Immobilon P membrane (Millipore Corp.) as described previously (8Bregman D.B. Battacharya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Abstract Full Text PDF PubMed Google Scholar). Blots were blocked, incubated with affinity-purified IgGs directed against DAKAP550 (1:2000, relative to serum), and washed as described previously (11Glantz S.B. Li Y. Rubin C.S. J. Biol. Chem. 1993; 268: 12796-12804Abstract Full Text PDF PubMed Google Scholar, 33Hirsch A.H. Glantz S.B. Li Y. You Y. Rubin C.S. J. Biol. Chem. 1992; 267: 2131-2134Abstract Full Text PDF PubMed Google Scholar). Antigen-IgG complexes were visualized by an indirect chemiluminescence procedure (28Ndubuka C. Li Y. Rubin C.S. J. Biol. Chem. 1993; 268: 7621-7624Abstract Full Text PDF PubMed Google Scholar, 33Hirsch A.H. Glantz S.B. Li Y. You Y. Rubin C.S. J. Biol. Chem. 1992; 267: 2131-2134Abstract Full Text PDF PubMed Google Scholar). Signals were recorded on Kodak XAR-5 x-ray film. No signals were observed when Western blots were probed with antibodies in the presence of 5 μg/ml fu-A or fu-B protein (see above). The Canton S strain was used for all experiments. A mutant line (DfC1) JC70, which is deficient in the X chromosome region 4C15-16; 5A1-2, was obtained from the Bowling GreenDrosophila Stock Center (Bowling Green, OH).Drosophila carrying ethyl methanesulfonate-induced mutations in the 4F1.2 region of the X chromosome (fs(1)456v24, fs(1) M60) were generously provided by Dr. N. Perrimon (Department of Genetics, Harvard Medical School) and Dr. R. Nagoshi (Department of Biology, University of Iowa). Embryos (0–24 h) were collected, dechorionated, and fixed according to the procedure of Mitchison and Sedat (34Mitchison T.J. Sedat J. Dev. Biol. 1983; 99: 261-264Crossref PubMed Scopus (158) Google Scholar). Specimens were blocked, washed, and then incubated with anti-DAKAP550 IgGs for 16 h at 4 °C in 10 mm sodium phosphate buffer, pH 7.4, containing 0.15m NaCl (phosphate-buffered saline), 20% fetal bovine serum, and 0.1% (v/v) Tween 20 as described previously (12Glantz S.B. Amat J.A. Rubin C.S. Mol. Biol. Cell. 1992; 3: 1215-1228Crossref PubMed Scopus (108) Google Scholar). Affinity-purified anti-DAKAP IgGs were used at 1:160 dilution (relative to serum). After extensive washing with phosphate-buffered saline, 0.1% Tween 20, samples were incubated for 2 h at 20 °C with horseradish peroxidase-coupled sheep IgGs directed against rabbit immunoglobulins (Bio-Rad) in the buffer specified above. DAKAP550·IgG complexes were detected by incubation of the specimens with diaminobenzidine and H2O2 (12Glantz S.B. Amat J.A. Rubin C.S. Mol. Biol. Cell. 1992; 3: 1215-1228Crossref PubMed Scopus (108) Google Scholar). In the presence of these substrates, peroxidase catalyzes the synthesis of an insoluble reaction product that is observed as a dark precipitate by light microscopy. To determine whether Drosophila expresses AKAPs, cytosolic and particulate proteins from fly embryos were size-fractionated by denaturing electrophoresis, transferred to a membrane, and incubated with 32P-labeled bovine RIIβ. In this “overlay assay” binding sites in immobilized AKAPs renature and sequester radiolabeled RII subunits (8Bregman D.B. Battacharya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Abstract Full Text PDF PubMed Google Scholar, 9Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Abstract Full Text PDF PubMed Google Scholar). Autoradiography revealed a group of cytosolic RII-binding proteins with molecular weights ranging from 70,000 to >200,000 (Fig.1, lane 2). Candidate cDNA clones for Drosophila AKAPs (DAKAPs) were retrieved from an embryo expression library (in bacteriophage λZAP) by using a modification of the overlay assay (8Bregman D.B. Battacharya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Abstract Full Text PDF PubMed Google Scholar). Five cross-hybridizing clones were isolated (Fig. 2). After recombinant pBluescript phagemids were excised from λZAP (24Short J.M. Sorge J.A. Methods Enzymol. 1992; 216: 495-508Crossref PubMed Scopus (63) Google Scholar), nested deletions of the cDNAs were prepared by exonuclease III digestion and religation. Sequencing of these overlapping cDNAs yielded an open reading frame of 3.2 kbp. To obtain contiguous flanking cDNA, a 5′-stretched embryonic cDNA expression library (λgt11) was screened for β-galactosidase fusion proteins that bound32P-RIIβ with high affinity. A cDNA named clone Z was obtained (Fig. 2). Subsequently, a 5′-terminalEcoRI-NcoI fragment (330 bp) from clone Z was employed as a template for synthesis of random-primed,32P-labeled cDNA. This probe was used to re-screen the λgt11 library via DNA hybridization. Four of 13 positive recombinant phage contained cDNAs that extended the 5′ end of the open reading frame (clones II-1, II-2, II-5, and II-7, Fig.2). Comparison of sequences for clones II-1 and II-7 revealed a 618-bp deletion in the latter cDNA (Fig. 2). This is probably due to alternative retention/excision of an exon because sequences at the 5′ and 3′ ends of the DNA fragment deleted in clone II-7 correspond to consensus splice donor and acceptor sites. Alternative splicing was not detected at other sites in DAKAP cDNAs. Exhaustive screening of available Drosophila cDNA libraries failed to yield clones containing contiguous segments of upstream or downstream cDNA. Sequencing of fragments of the DAKAP gene enabled extension of the coding region ∼900 bp downstream. Unfortunately, the presence of very long introns at the 5′ and 3′ ends of the DAKAP gene have impeded determination of the translation initiation and termination codons. Nevertheless, a composite 7.1-kbp DNA coding region has been established for a novel, invertebrate PKA-binding protein by assembling overlapping DNA sequences. Each segment of DNA was sequenced at le
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