Discovery and characterization of a Caenorhabditis elegans anchor protein (AKAP(CE)) that binds protein kinase AI
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Classical A kinase anchor proteins (AKAPs) preferentially tether type II protein kinase A (PKAII) isoforms to sites in cytoskeleton and organelles. It is not known if distinct proteins selectively sequester regulatory (R) subunits of type I PKAs, thereby diversifying functions of these critical enzymes. In C. elegans, a single type I PKA mediates all aspects of cAMP signaling. I have discovered a cDNA that encodes a binding protein (AKAPCE) for the regulatory subunit (RCE ) of C. elegans PKAICE. AKAPCE is a novel, highly-acidic, RING finger protein composed of 1,280 amino acids. It binds RI-like RCE with high affinity and neither RIIalpha nor RIIbeta competitively inhibits formation of AKAPCE·R CE complexes. The RCE binding site was mapped to a segment of 20 amino acids in an N terminal region of AKAPCE. Three large aliphatic amino acids (L236, I248, L 252) in the tethering domain of AKAPCE (residues 236-255) are crucial for ligation of RCE. Their sidechains apparently generate a precisely-configured hydrophobic binding pocket that accommodates an apolar surface on RCE dimers. Basic residues (H254R 255K256) at the C terminus of the tethering site set an upper limit on affinity for RCE. A central dipeptide (F 243S244) contributes critical and distinctive properties of the tethering site. S244 is essential for selective binding of RCE and exclusion of RII isoforms. The aromatic hydrophobic character of F243 ensures maximal RCE binding activity, thereby supporting a "gatekeeper" function of S244. Substitution of F243S244 with LV generated an RII-specific AKAP. RCE and RII subunits contain similar dimerization domains. AKAP-binding domains of RCE (residues 23-47) and RII differ markedly in size, amino acid sequence and docking specificity. Four hydrophobic residues (C23, V27, I32, C 44) in RCE are crucial for avid binding with AKAP CE, whereas sidechains from L20, L35, V36, I40 and I14 have little impact on complex formation. Y26 is embedded in the docking domain, but its aromatic ring is required for RCE-RCE dimerization. Residues 236-255 in AKAPCE also constitute a binding site for mammalian RIalpha. RIalpha (PKAIalpha) is tightly sequestered by AKAPCE in vitro (KD∼10 nM) and in the environment of intact cells. AKAPCE·RCE and AKAPCE·RIalpha complexes accumulate in intact cells. Thus, the tethering domain of AKAPCE provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.
Source: Dissertation Abstracts International, Volume: 61-09, Section: B, page: 4559.;Advisors: Charles S. Rubin.