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

Abstract

Caenorhabditis elegans protein kinase A (PKAI CE) (5,6,9,10). Clustering of a high concentration of AKAP⅐PKAII complexes in proximity with PKA substrate/effector proteins in cytoskeleton/organelles enables efficient reception, rapid amplification, and precisely focused targeting of cAMP signals. Consequently, PKA-catalyzed phosphorylation of co-localized effector proteins is optimized (11-13). Key tenets of the preceding signaling model have been verified. Disruption of AKAP⅐PKA complexes in situ markedly diminishes the ability of cAMP to regulate such critical physiological processes as ion transport, gene transcription, apoptosis, and hormone secretion (8,(13)(14)(15)(16).Mammals employ Ͼ20 distinct AKAPs to adapt type II PKAs for specialized functions (5)(6)(7)(8)17). Amino acid sequences of the anchor proteins are markedly divergent; thus, AKAPs are functional, not structural, homologs. Different AKAPs accumulate in distinct locations; examples include cytoplasmic surfaces of plasma membrane, mitochondria, Golgi membranes, and centrosomes (5,6,9,10,18). Routing of PKAII to specific microenvironments is governed by unique targeting/anchoring domains in individual AKAPs (9, 19 -22). Primary structures of RII-binding sites in AKAPs are not highly conserved (e.g. see alignments in Refs. 23 and 24). However, the folding pattern and physical properties of amino acids that subserve the ligation of RII subunits are universal properties of AKAPs. The RII-binding region of typical AKAPs comprises 16 -20 contiguous amino acids that have a predicted propensity to fold into an amphipathic ␣-helix (25). The ␣-helix includes six critical, precisely positioned amino acids whose large aliphatic side chains co-operatively create an extended hydrophobic surface (19). The size, hydrophobicity, and configuration of this domain generate a unique receptor site for a complementary, apolar docking surface that is produced from the folding of ϳ30 amino acid residues in RII␣ or RII␤ dimers (26 -29). Robust hydrophobic * This work was supported by National Institutes of Health Grant GM57660 and a stipend from Training Grant GM07620 (to R. A. A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed: Dept.

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

“…NMJ localization is specific for RI␣ because the corresponding docking region of RII␣ (residues 1-44) does not confer accumulation. The localization of RI␣ is affected by mutation of the conserved residues Val-22, Ile-27, and Phe-54, which are essential for the interaction of R CE with AKAP CE (11) and for RI␣ binding to D-AKAP1 in vitro (13). In fact, we observed an enrichment of D-AKAP1 at the NMJ, implying that RI␣ could be recruited by this anchoring protein.…”

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

“…AKAP CE (239-248), with Ser at position 6, did not, however, have similar affinities for RI and RII as expected by the rule, but interacted only with RI or RI-like subunits (20). BIG2 domain C (525-539), which interacted with both RI␣ and RII, has Phe, a hydrophobic amino acid with an aromatic side chain at position 6, adjacent to Glu-531 (position 7), which may or may not serve as ''gatekeeper'' to form a binding pocket accommodating a portion of the R docking domain in a manner similar to that of Phe-243 and Ser-244 in AKAP CE (20). Further mutagenesis and structural experiments are needed to resolve the question.…”

“…Domain A (34-48), with Ala in position 6, interacted with RI␣ and RI␤, but not RII␣ or RII␤, whereas domain B, BIG2 (284-298), with Val in position 6, interacted with RII rather than RI subunits, which seems to fit a ''three amino acid'' rule (29). AKAP CE (239-248), with Ser at position 6, did not, however, have similar affinities for RI and RII as expected by the rule, but interacted only with RI or RI-like subunits (20). BIG2 domain C (525-539), which interacted with both RI␣ and RII, has Phe, a hydrophobic amino acid with an aromatic side chain at position 6, adjacent to Glu-531 (position 7), which may or may not serve as ''gatekeeper'' to form a binding pocket accommodating a portion of the R docking domain in a manner similar to that of Phe-243 and Ser-244 in AKAP CE (20).…”

“…Most analyses of PKA anchoring have focused on AKAPs that interact with RII, especially RII␣ (9,18). So-called dual AKAPs, such as D-AKAP1 and D-AKAP2, can interact with both RI and RII (14,15), and there are several AKAPs that associate specifically with RI or RI-like subunits, such as AKAP CE (19,20). RI␤ has been implicated in the presynaptic regulation of hippocampal LTP͞LTD and in vivo with synaptic plasticity in the visual cortex (21-23), but no AKAP for RI␤ was identified in those systems.…”

“…RI␤ has been implicated in the presynaptic regulation of hippocampal LTP͞LTD and in vivo with synaptic plasticity in the visual cortex (21-23), but no AKAP for RI␤ was identified in those systems. Three positions that are identical in RI␣ (Val-22, Ile-27, and Cys-39) and RI CE (Val-27, Ile-32, and Cys-44) govern their interaction with DAKAP1 and AKAP CE (17,20), respectively. Presence of the same residues in RI␤ (Val-22, Ile-27, and Cys-39) presumably provides a structural basis for AKAP interactions.…”