Generation of a Novel A Kinase Anchor Protein and a Myristoylated Alanine-rich C Kinase Substrate-like Analog from a Single Gene

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 ϳ3.8) 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, ⌬DAKAP200. ⌬DAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. ⌬DAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and ⌬DAKAP200 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; ⌬DAKAP200 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.Cyclic AMP-dependent protein kinases (PKAs) 1 mediate actions of hormones that stimulate adenylate cyclase (1-4). Signals carried by cAMP often regulate activities of proteins that accumulate at discrete intracellular locations (e.g. ion channels in plasma membrane) (5-8). A kinase anchor proteins (AKAPs) guide transmission and routing of cAMP signals to such microenvironments. AKAPs have an avid binding site for regulatory subunits (RII) of PKAII isoforms 2 and distinct domains that target the tethered PKA holoenzyme to docking sites in organelles or cytoskeleton (5, 6, 9, 10). Accumulation of a high concentration of anchored PKAII in proximity with clustered substrate-effector protein promotes efficient reception, amplification, and precisely focused transmission of signals borne by cAMP (5-8).Several fundamental questions about AKAP⅐RII complexes remain unresolved. Current models suggest that AKAPs statically associate with docking sites in organelles. However, it is possible that AKAP⅐RII complexes shuttle dynamically between intracellular loci. This proposition has neither been excluded nor systematically investigated. Many physiological processes are coordinately regulated by groups of hormones. Homeostatic control is often achieved through synergistic or antagonistic interactions among signaling pathways that are activated by different second messenger molecules. A key question is how signals transmitted via AKAP⅐PKAII complexes are integrated with inputs from pathways controlled by other second messengers in order to regulate common effector proteins. PKA anchoring may be regulated by pretranslat...

Characterization of Structural Features That Mediate the Tethering of Caenorhabditis elegans Protein Kinase A to a Novel A Kinase Anchor Protein

“…It is proposed that Ala is required for selective tethering of RI␣; Ser enables dual binding of RI␣ and RII␣ with similar affinities, and Ile, Val, or Leu promotes RII binding. The last suggestion is consistent with previously published alignments and conclusions indicating that Leu, Ile, and Val invariably occupy these positions in RII-binding proteins (9,23,24,37,52), and mutation of these residues to Ala compromises RII binding activity in all cases (19,37,52,59). Mutagenesis/expression studies on AKAP CE document Ser 244 or Val 244 (corresponding to position 6) are crucial determinants for R isoform selectivity.…”

Characterization of the Targeting, Binding, and Phosphorylation Site Domains of an A Kinase Anchor Protein and a Myristoylated Alanine-rich C Kinase Substrate-like Analog That Are Encoded by a Single Gene

“…PSD modules (ϳ25-30 residues) of vertebrate myristoylated alanine rich C kinase substrate (MARCKS) proteins bind calcium-calmodulin, phospholipids, and actin; cooperate with N-terminal myristate to attach proteins to cell membranes; constitute specific target sites for protein kinase C (PKC)-catalyzed phosphorylation; and participate in dynamic regulation of the actin cytoskeleton that is associated with such fundamental cellular processes as motility and differentiation (7)(8)(9)(10)(11)(12). Thus, predicted properties of DAKAP200 suggest that this polypeptide is polyfunctional, differentially targeted to membranes, and involved in routing and integrating signals propagated by three important second messengers: cAMP, diacylglycerol, and calcium (6). Numerous caveats preclude acceptance of these ideas solely on the basis of information obtained from the amino acid sequence of DAKAP200.…”

“…The discovery (6) of a novel cDNA and unique gene that encode Drosophila A kinase anchor protein 200 (DAKAP200) 1 provides an opportunity to investigate molecular mechanisms underlying integrated responses to multiple second messengers. DAKAP200 avidly binds regulatory subunits (RII) of protein kinase AII (PKAII) isoforms in vitro and in cells (6). The amino acid sequence of this 753-residue PKAII anchor protein also includes an Nterminal myristoylation motif and a potential "phosphorylation site domain" (PSD) (6).…”

“…DAKAP200 avidly binds regulatory subunits (RII) of protein kinase AII (PKAII) isoforms in vitro and in cells (6). The amino acid sequence of this 753-residue PKAII anchor protein also includes an Nterminal myristoylation motif and a potential "phosphorylation site domain" (PSD) (6). PSD modules (ϳ25-30 residues) of vertebrate myristoylated alanine rich C kinase substrate (MARCKS) proteins bind calcium-calmodulin, phospholipids, and actin; cooperate with N-terminal myristate to attach proteins to cell membranes; constitute specific target sites for protein kinase C (PKC)-catalyzed phosphorylation; and participate in dynamic regulation of the actin cytoskeleton that is associated with such fundamental cellular processes as motility and differentiation (7)(8)(9)(10)(11)(12).…”

A screen for modifiers of RacGAP(84C) gain-of-function in theDrosophilaeye revealed the LIM kinase Cdi/TESK1 as a downstream effector of Rac1 during spermatogenesis

“…MESR2/AKAP200 encodes the Drosophila A kinase anchor protein, AKAP200, with a molecular mass of 200 kDa. AKAP200 binds the cyclic AMP-dependent protein kinase A (PKA) that has a non-uniform distribution in cells (Li et al, 1999). We previously demonstrated that overexpression of RacGAP(84C) perturbs the sub-cortical localisation of polymerised actin in embryonic cells (Guichard et al, 1997;Raymond et al, 2001), which would presumably prevent the normal localisation of AKAP200 with respect to the cytoskeleton.…”