A kinase anchor protein 200

Protein Interactions

An approximate location for the PKAII tethering site in Akap200 was established by sequencing five cDNAs that directed synthesis of RII-binding, fusion proteins in plaques of a bacteriophage expression library. The sequence of the smallest cDNA (147 bp) encodes a region of the anchor protein bounded by Asp⁴⁹⁰ and Glu⁵³⁸. The same sequence was present in each of the larger (1.1-1.7 kbp) cDNAs. Tethering sites in mammalian AKAPs are composed of ~20 amino acids and contain a precisely spaced group of residues with large, aliphatic side chains (Leu, Val, Ile, and Thr) that cooperatively govern sequestration of RIIa and RIIß subunits. Residues 511-530 in Akap200 can be aligned with tethering regions of mammalian AKAPs, so that Ile⁵¹¹, Ile⁵¹⁸, Val⁵¹⁹, Thr⁵²³, and Val⁵³⁰ are in register with essential hydrophobic residues in previously characterized RII binding sites. A final conserved position is occupied by a smaller hydrophobic residue (Ala⁵²⁷) in the Drosophila anchor protein. RII binding domains of mammalian AKAPs are predicted to fold as an amphipathic a-helix that contains one markedly hydrophobic surface. This feature is also evident in the segment of Akap200 that encompasses residues 511-528. Folding algorithms predict that this partial polypeptide is organized into an a-helix in which 9 of 10 side chains on one surface have hydrophobic character. Thus, amino acids 511-530 constitute a candidate RII binding site in Akap200 (Li, 1999).

To further characterize the tethering domain, a fragment of Akap200 cDNA (nucleotides 1577-2314) that encodes amino acids 475-753 was amplified by polymerase chain reaction. The soluble His tagged fusion protein (named p-DAKAP70) was purified to near homogeneity by affinity chromatography on Ni²⁺-chelate Sepharose 4B resin. Small amounts of immobilized p-DAKAP70 avidly bind ³²P-RIIß (human) in an overlay assay performed with a low concentration of labeled ligand. Similar results were obtained with radiolabeled murine RIIa, thereby indicating that the tethering domain of Akap200 complexes both RII isoforms with high affinity. A caveat is that both binding studies and functional screening of cDNA expression libraries were designed and executed on the basis of a logical but unproved assumption, that mammalian RII isoforms are interchangeable substitutes for authentic Drosophila RII (RII_DR) subunits (Li, 1999).

To verify this assumption and demonstrate more directly the physiological relevance of the novel fly anchor protein, it was essential to examine the ability of Akap200 to bind RII_DR. The gene encoding the 376-residue RII_DR polypeptide has been cloned and sequenced. Structural features that govern subunit dimerization and create a docking surface for AKAPs are located near the amino terminus (residues 1-50) of mammalian RII isoforms. Alignment of the N termini of RII_DR and RIIa reveals that the two sequences are quite divergent (only 44% identity). However, groups of aromatic residues (Phe and Tyr) and amino acids with large aliphatic side chains (Leu, Val, and Ile) contribute the essential functional properties of dimerization and AKAP binding regions in RIIa and RII. Amino acids with these characteristics are conserved at all corresponding positions within residues 1-50 of RII _DR (Li, 1999)

Like mammalian RII subunits, RII_DR contains the PKA phosphorylation site sequence (RRXSX) in a linker region between the dimerization-AKAP binding domain and the cAMP binding sites. Thus, RII_DR was labeled by incubation with Mg-gamma-³²P ATP and the catalytic (C) subunit of PKA. ³²P-Labeled RII_DR binds with low levels of p-DAKAP70 and also forms a stable complex with bovine AKAP75. Thus, structural features that mediate interactions between RII subunits and AKAPs have co-evolved and are conserved from flies to humans. Binding interactions between RIIa, RIIß, or RII_DR and various AKAPs are sufficiently similar to enable their interchangeable use. Since recombinant mammalian RII isoforms are available in plentiful supply and these proteins are more thoroughly characterized than RII_DR, they were employed for most of the studies presented in this study. Repetition of experiments with RII_DR yielded similar results in all instances (Li, 1999).

The physiological relevance of Akap200 was investigated by testing the ability of the anchor protein to bind an endogenous ligand, RII_DR. Several domains in Akap200 may contribute to the targeting of tethered PKAII. Two Pro-rich regions (residues 328-332 and 468-479) are potential binding sites for cytoskeleton/organelle-associated proteins that contain Src homology 3 domains. Amino acids 2-7 constitute an acceptor site for N-myristoyltransferase. Myristoylation of Akap200 would provide a long saturated aliphatic chain that inserts into the hydrocarbon interior of phospholipid bilayers. A segment of Akap200 (residues 118-148) includes a PSD-like cluster of 13 Lys, five Ser, and five large hydrophobic residues. Positive charge in PSD2S promotes electrostatic binding with negatively charged head groups of membrane phospholipids. Intercalation of PSD hydrophobic side chains into the apolar interior of a bilayer further stabilizes association of PSD-containing proteins with membranes. N-terminal myristate and a PSD are critical features of MARCKS proteins, which mediate interactions between plasma membrane and F-actin. The MARCKS PSD is phosphorylated in situ by diacylglycerol-activated protein kinase C isoforms. Phosphorylation inhibits binding with membrane phospholipids, enables translocation of MARCKS from cell surface to cytoplasm, and promotes cytoskeleton remodeling. The nonphosphorylated PSD sequesters calmodulin in a calcium-dependent manner. Binding of Ca²⁺-calmodulin diminishes the ability of MARCKS to cross-link and bundle actin filaments. By analogy, the fly anchor protein may be involved in integrating signals propagated by three critical second messenger molecules: cAMP, diacylglycerol, and calcium. Moreover, the PSD region of Akap200 may enable phosphorylation-controlled shuttling of tethered PKA between two or more intracellular sites (Li, 1999).

Drosophila A kinase anchor protein 200 (Akap200), is predicted to be involved in routing, mediating, and integrating signals carried by cAMP, Ca²⁺, and diacylglycerol. Experiments designed to assess this hypothesis establish (1) the function, boundaries and identity of critical amino acids of the protein kinase AII (PKAII) tethering site of Akap200; (2) demonstrate that residues 119-148 mediate binding with Ca²⁺-calmodulin and F-actin; (3) show that a polybasic region of Akap200 is a substrate for protein kinase C; (4) reveal that phosphorylation of the polybasic domain regulates affinity for F-actin and Ca²⁺-calmodulin, and (5) indicate that Akap200 is myristoylated and that this modification promotes targeting of Akap200 to plasma membrane. DAkap200, a second product of the Akap200 gene, cannot tether PKAII. However, DAkap200 is myristoylated and contains a phosphorylation site domain that binds Ca²⁺-calmodulin and F-actin. An atypical amino acid composition, a high level of negative charge, exceptional thermostability, unusual hydrodynamic properties, properties of the phosphorylation site domain, and a calculated M_r of 38,000 suggest that DAkap200 is a new member of the myristoylated alanine-rich C kinase substrate protein family. Akap200 is a potentially mobile, chimeric A kinase anchor protein-myristoylated alanine-rich C kinase substrate protein that may facilitate localized reception and targeted transmission of signals carried by cAMP, Ca²⁺, and diacylglycerol (Rossi, 1999).

The observation that amino acids 2-7 constitute a potential myristoylation site resulted in identification of a targeting domain. Metabolic labeling with [³H]myristate and immunoprecipitation with anti-Akap200 IgGs has revealed that endogenous Akap200 and DAkap200 are myristoylated in situ in Drosophila S2 cells. Incorporated myristate provides a long alkyl chain that inserts into the interior of phospholipid bilayers, thereby promoting the targeting, anchoring, and enrichment of Akap200 at a membrane surface. Myristoylated anchor protein is also synthesized in mammalian (AV12) cells that contain a Akap200 transgene. A deleted version of Akap200 that lacks residues 1-7 was expressed in AV12 cells, but the protein failed to incorporate [³H]myristate. Thus, the N-terminal region of Akap200 is the sole site of myristoylation and a probable targeting domain for the anchor protein. Several myristoylated signaling proteins contain proximal Cys residues that undergo palmitoylation. Acylation at multiple sites causes extremely stable membrane anchoring. However, Akap200 is not modified by palmitoylation (Rossi, 1999).

N-terminal myristoylation is essential for routing various signaling proteins, including Src and MARCKS, to plasma membrane. Mutations that prevent myristoylation elicit mislocalization and loss of biological function. Myristoylated MARCKS accumulates at focal adhesions or at the junction of cortical cytoskeleton and plasma membrane. MARCKS may simultaneously bind the lipid bilayer (via myristate) and the F-actin-based cytoskeleton. In this configuration, MARCKS mediates interactions between the membrane and actin filaments that stabilize the cytoskeleton. Nonmyristoylated MARCKS is restricted to cytoplasm, and its ability to modulate cytoskeleton organization is abrogated (Rossi, 1999 and references therein).

Akap200 and DAkap200 are specifically enriched in the plasma membrane of Drosophila S2 cells. Epitope-tagged wild type Akap200 is also differentially targeted to plasma membrane in transfected S2 cells. Tagged nonmyristoylated Akap200 is evenly distributed throughout the cell cytoplasm. Thus, N-terminal myristoylation is essential for organelle-specific accumulation of Akap200·RII (PKAII) complexes. Mammalian AKAP18 contains a classical RII tethering site, but docking of this anchor protein with plasma membrane is guided by three acylated amino acids (Rossi, 1999).

Gly¹ is myristoylated, whereas Cys³ and Cys⁴ are palmitoylated. Deletion or mutation of Gly¹ abrogates myristoylation but has no effect on AKAP18 localization. Likewise, mutation of Cys³ or Cys⁴ alone does not alter association of AKAP18 with plasma membrane. However, elimination of all acylation sites yields an AKAP18 variant that accumulates in cytoplasm. Thus, myristoylation of AKAP18 supports, but is not essential for, targeting/anchoring of PKAII (Rossi, 1999 and references therein).

Deletion mutagenesis mapped the Akap200 RII binding domain to a region that includes amino acids 511-531. Activity and selectivity of this PKAII tethering site were not dependent on the context of flanking N- and C-terminal sequences. Conserved hydrophobic residues in the tethering region are predicted to generate an RII binding surface comparable in size and shape with that produced from the corresponding region of mammalian AKAP75 and AKAP79. Substitution of Ile⁵¹⁸ and Val⁵¹⁹ or Thr⁵²³ with Ala sharply diminish RII ligation. This is interpreted as a consequence of the disruption of two parameters (size and shape) in a structure based on cooperative interactions among large, aliphatic side chains. Substitution of Ala⁵¹⁵ with Ser reduced RII binding activity by ~70%, thereby indicating that a hydrophobic side chain at this position is essential for a maximal ligand affinity. This mutation reveals evolutionary divergence in the fly AKAP. The analogous Ala to Ser mutation has no effect on RII sequestration by mammalian AKAP-KL (Rossi, 1999 and references therein).

A PSD (residues 119-148) from Akap200/DAkap200 binds F-actin and Ca²⁺-calmodulin. Ser¹³² and other serines (Ser¹³⁵ and/or Ser¹³⁷) in the PSD region are phosphorylated by PKC. Both calmodulin and actin binding activities are lost as a consequence of PSD-directed phosphorylation. This provides a mechanism for reversibly altering the location and Ca²⁺-calmodulin-related effector functions of Akap200 and DAkap200. The physiological importance of multifunctional PSDs in vertebrate MARCKS and MARCKS-like proteins is paralleled by a high degree of sequence conservation across species. However, alignment of PSD sequences disclosed that the Akap200 PSD is a novel and rather divergent member of this domain family. A distinctive feature of the Drosophila PSD is the compact central cluster of large hydrophobic residues (Trp¹³¹, Phe¹³³, Ile¹³⁶, Phe¹³⁸) that intermingle with three Ser residues that are PKC targets. This arrangement involves a contiguous block of only eight amino acids. Introduction of a high level of negative charge adjacent to sites where Trp, Phe, and Ile side chains are immersed in a lipid bilayer may negate membrane association by electrostatic repulsion from negatively charged phospholipid head groups. This 'electrostatic switch mechanism' may be less efficient and require a higher level of phosphorylation in vertebrate PSDs where serines and hydrophobic amino acids are dispersed among a total of 14 residues. Incorporation of a novel PSD into Akap200 produces a multifunctional anchor protein with enhanced capacities for intracellular targeting, regulation via phosphorylation, and integration of multiple second messenger signals (Rossi, 1999).

MARCKS is a major PKC substrate in many mammalian cells, where it mediates aspects of cytoskeleton dynamics. Incorporation of an RII binding site within a PKC effector protein enables a novel mode of cross-talk between two signal transduction pathways. PKC-catalyzed phosphorylation of the Akap200 PSD could elicit translocation of anchor protein·PKAII complexes from plasma membrane/cortical cytoskeleton to cytoplasm (or elsewhere). Consequently, hormones that stimulate synthesis of lipid second messengers could determine intracellular target sites at which the cAMP/PKA signaling pathway exerts its actions. Phosphorylation-dependent targeting of Akap200·RII complexes could constitute a novel, reversible mode of routing signals carried by cAMP. Previously characterized AKAPs appear to engage in static anchoring of PKAII isoforms (Rossi, 1999 and references therein).

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