Characterization of Structural Features That Mediate the Tethering of Caenorhabditis elegans Protein Kinase A to a Novel A Kinase Anchor Protein: INSIGHTS INTO THE ANCHORING OF PKAI ISOFORMS

Robert G Angelo,Charles S Rubin

doi:10.1074/jbc.275.6.4351

Robert G Angelo, Charles S Rubin

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https://doi.org/10.1074/jbc.275.6.4351

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Caenorhabditis elegans protein kinase A (PKAI(CE)) is tethered to organelles in vivo. A unique A kinase anchor protein (AKAP(CE)) avidly binds the RI-like regulatory subunits (R(CE)) of PKAI(CE) and stringently discriminates against RIIalpha and RIIbeta subunits, the preferred ligands for classical AKAPs. We elucidated structural features that stabilize AKAP(CE).R(CE) complexes and confer atypical R isoform specificity on the anchor protein. Three large aliphatic amino acids (Leu(236), Ile(248), and Leu(252)) in the tethering domain of AKAP(CE) (residues 236-255) are crucial for ligation of R(CE). Their side chains apparently generate a precisely configured hydrophobic binding pocket that accommodates an apolar surface on R(CE) dimers. Basic residues (His(254)-Arg(255)-Lys(256)) at the C terminus of the tethering site set an upper limit on affinity for R(CE.) A central dipeptide (Phe(243)-Ser(244)) contributes critical and distinctive properties of the tethering site. Ser(244) is essential for selective binding of R(CE) and exclusion of RII isoforms. The aromatic hydrophobic character of Phe(243) ensures maximal R(CE) binding activity, thereby supporting a "gatekeeper" function of Ser(244). Substitution of Phe(243)-Ser(244) with Leu-Val generated an RII-specific AKAP. R(CE) and RII subunits contain similar dimerization domains. AKAP-binding domains of R(CE) (residues 23-47) and RII differ markedly in size, amino acid sequence, and docking specificity. Four hydrophobic residues (Cys(23), Val(27), Ile(32), and Cys(44)) in R(CE) are crucial for avid binding with AKAP(CE), whereas side chains from Leu(20), Leu(35), Val(36), Ile(40), and Ile(41) have little impact on complex formation. Tyr(26) is embedded in the docking domain, but its aromatic ring is required for R(CE)-R(CE) dimerization. Residues 236-255 in AKAP(CE) also constitute a binding site for mammalian RIalpha. RIalpha (PKAIalpha) is tightly sequestered by AKAP(CE) in vitro (K(D) = approximately 10 nM) and in the environment of intact cells. The tethering domain of AKAP(CE) provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.

Highlights

Mammals employ Ͼ20 distinct A kinase anchor proteins (AKAPs) to adapt type II protein kinases (PKAs) for specialized functions [5,6,7,8, 17]
To guide studies on individual amino acids that are crucial for the ligation of RCE, the sequence of the tethering region of AKAPCE was aligned with a prototype mammalian RII-binding domain
236 –255 in AKAPCE are predicted to fold into an ␣-helix that contains opposing hydrophobic and hydrophilic surfaces (Fig. 1B)

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Introduction

Mammals employ Ͼ20 distinct AKAPs to adapt type II PKAs for specialized functions [5,6,7,8, 17]. RCE polypeptides that include single or double Cys to Ala mutations (Ala23 and Ala44) dimerize normally (Fig. 4).3 the amounts of wild type and mutant RCE proteins complexed by the AKAPCE tethering domain were not altered when 0.1 M dithiothreitol was added to the binding buffer.3 The indicated concentration of dithiothreitol reduces all Cys residues in native R subunit dimers [55].

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