Molecular Characterization of a cDNA That Encodes Six Isoforms of a Novel Murine A Kinase Anchor Protein

Abstract

We have cloned cDNA that encodes six novel A kinase anchor proteins (collectively named AKAP-KL). AKAP-KL diversity is generated by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL polypeptides are evident in lung, kidney, and cerebellum, but are absent from many tissues. Different isoforms predominate in different tissues. Thus, AKAP-KL expression is differentially regulated in vivo. All AKAP-KL isoforms contain a 20-residue domain that avidly binds (K d ϳ 10 nM) regulatory subunits (RII) of protein kinase AII and is highly homologous with the RII tethering site in neuronal AKAP75. The distribution of AKAP-KL is strikingly asymmetric (polarized) in situ. Anchor protein accumulates near the inner, apical surface of highly polarized epithelium in tubules of nephrons. Both RII and AKAP-KL are enriched at an intracellular site that lies just below the plasma membrane of alveolar epithelial cells in lung. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells. We also demonstrate that the tethering domain of AKAP-KL avidly ligates RII subunits in intact cells. AKAP-KL may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors to capture, amplify, and precisely focus diffuse, trans-cellular signals carried by cAMP. Protein kinase A (PKA)1 mediates actions of hormones and neurotransmitters that activate adenylate cyclase (1-4). Signals carried by cAMP are often directed to effectors that accumulate at discrete intracellular sites (5-7). Targeting of signals to these sites can be achieved by generating a non-uniform distribution of PKA molecules. This occurs when PKAII␣ and II␤ isoforms are attached to cytoskeleton or organelles by A kinase anchor proteins (AKAPs) (5, 6). Prototypic anchor proteins (AKAPs 75, 79, and 150) have a binding site for regulatory (RII) subunits of PKAII isoforms and distinct domains that mediate non-covalent coupling of AKAP⅐PKAII complexes to the microtubule-based dendritic cytoskeleton of neurons and the cortical actin cytoskeleton of non-neuronal cells (5-13). Both cytoskeletal locations are closely apposed to the plasma membrane. Thus, anchored PKAII is positioned near a signal generator (adenylate cyclase) and multiple PKA substrate/effector proteins (e.g. myosin light chain kinase, microtubuleassociated protein-2, ion channels, serpentine receptors that couple with G s ) (5,6,14). Effector proteins in cAMP signaling pathways can also be associated with organelles, specialized regions of plasma membrane, or sites in cytoskeleton that are separated from adenylate cyclase by substantial distances. Novel RII-binding proteins apparently participate in the assembly of "distal signaling modules" that are associated with mitochondria, Golgi membranes, peroxisomes, and centrioles (5, 6, 15, 16).Anchored PKAII isoforms may be essential for dissemination of cAMP signals in highly polarized epithelium. Epithelial cells of l...

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

“…Three important determinants were discerned in these regions and rules for RI-selective, RII-selective and dual binding specificities were suggested. Our current results and previous studies (9,19,23,37,52,59) are in partial accord with these suggestions. However, other aspects of our observations suggest distinctly different interpretations.…”

“…Classical RII-selective AKAPs contain a hydrophobic dipeptide composed exclusively of Leu, Val, or Ile residues (Leu-Val in AKAP75; Ile-Ile in S-AKAP84; Fig. 1A) that is crucial for the generation and maintenance of the RII-binding site (19,52). The appearance of a Phe 243 -Ser 244 dipeptide in the corresponding region of the AKAP CE tethering domain represents a major deviation from the previously studied paradigm.…”

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

“…2A) that cooperatively governs sequestration of RII␣ and RII␤ subunits (35). Residues 511-530 in DAKAP200 can be aligned with tethering regions of mammalian AKAPs (9,35,42) , and Val 530 are in register with essential hydrophobic residues in previously characterized RII binding sites ( Fig. 2A).…”

“…Binding of RII at tethering sites in AKAPs is governed by hydrophobic interactions (35,42). Structural features and RII binding assays suggest that amino acids 511-530 in DAKAP200 constitute an RII binding site.…”

Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2)

“…Because the antibodies that we used for both immunoblotting and immunoprecipitation were produced with a peptide representing amino acids 232-241 in BIG2, it is obvious that some forms of BIG2 proteins might have been missed in these experiments. Dong et al (35), for example, described six isoforms of AKAP-KL generated by alternative mRNA splicing. This could be an important mechanism for increasing diversity of BIG2 targeting and functional specificity.…”