Abstract
A unique Drosophila gene encodes two novel signaling proteins. Drosophila A kinase anchor protein 200 (DAKAP200) (753 amino acids) binds regulatory subunits of protein kinase AII (PKAII) isoforms in vitro and in intact cells. The acidic DAKAP200 polypeptide (pI approximately 3.8) contains an optimal N-terminal myristoylation site and a positively charged domain that resembles the multifunctional phosphorylation site domain of vertebrate myristoylated alanine-rich C kinase substrate proteins. The 15-kilobase pair DAKAP200 gene contains six exons and encodes a second protein, DeltaDAKAP200. DeltaDAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. DeltaDAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and DeltaDAKAP200 are evident in vivo at all stages of Drosophila development. Thus, both proteins may play important physiological roles throughout the life span of the organism. Nevertheless, DAKAP200 gene expression is regulated. Maximal levels of DAKAP200 are detected in the pupal phase of development; DeltaDAKAP200 content is elevated 7-fold in adult head (brain) relative to other body parts. Enhancement or suppression of exon 5 excision during DAKAP200 pre-mRNA processing provides potential mechanisms for regulating anchoring of PKAII and targeting of cAMP signals to effector sites in cytoskeleton and/or organelles.
Highlights
Cyclic AMP-dependent protein kinases (PKAs)1 mediate actions of hormones that stimulate adenylate cyclase [1,2,3,4]
A key question is how signals transmitted via AKAP1⁄7PKAII complexes are integrated with inputs from pathways controlled by other second messengers in order to regulate common effector proteins
To further the aim of elucidating the complete spectrum of A kinase anchor proteins (AKAPs) structure and function in Drosophila, we report on the characterization of a novel, multifunctional anchor protein named DAKAP200
Summary
Screening of cDNA Libraries—Expression libraries of Drosophila melanogaster (Canton S strain) cDNAs were searched for inserts encoding A kinase anchor proteins by functional (RII binding) assays and DNA hybridization. cDNA libraries were generated by reverse transcription of mRNAs isolated from embryos 0 –24 h after fertilization. After cloning the cDNA into pET14b, His-tagged RIIDR was expressed in E. coli and purified to near homogeneity by using the strategy described above for the partial DAKAP200 protein. The cDNA fragment was cloned into the Drosophila expression plasmid pMK33 HS (generously provided by Dr Nick Baker, Department of Molecular Genetics, Albert Einstein College of Medicine) that was cleaved with XhoI and SpeI. This placed the RIIDR cDNA downstream from a strong, copper(II)-inducible metallothionein promoter/enhancer and upstream from a polyadenylation signal. Amounts of protein in experimental samples were obtained from the linear portion of the standard curve
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