Mutagenesis of the regulatory subunit (RIIβ) of cAMP-dependent protein kinase IIβ reveals hydrophobic amino acids that are essential for RIIβ dimerization and/or anchoring RIIβ to the cytoskeleton

In neurons cAMP-dependent protein kinase IIβ (PKAIIβ) is sequestered in the dendritic cytoskeleton because the regulatory subunit (RIIβ) of the enzyme is tightly bound by A Kinase Anchor Proteins (AKAPs). The prototypic neuronal anchor protein AKAP75 has a COOH-terminal 22-residue RIIβ binding (tethering) site. A key feature of the tethering site is that several amino acids with large aliphatic side chains mediate the high-affinity binding of RIIβ. Mutagenesis, recombinant protein expression, and physicochemical characterization were used to investigate the structural basis for the homodimerization and AKAP75 binding activities of RIIβ. Several crucial residues are located in an NH₂-terminal region that encompasses amino acids 13-36. Substitution of Ala for Leu¹³ or Phe³⁶ generates monomeric RIIβ subunits that cannot bind AKAP75. The results are not due to general misfolding since mutant RIIβ monomers bind cAMP and inhibit the catalytic subunit of PKAIIβ with the same affinity and efficacy as wild-type RIIβ dimers. Moreover, substitution of Ala for Leu¹², Val²⁰, Leu²¹, Phe³¹, Leu³³, or Leu³⁹ and replacement of Leu¹³ with Ile or Val did not impair the dimerization reaction. Evidently, large hydrophobic side chains of Leu¹³ and Phe³⁶ play pivotal roles in stabilizing RIIβ-RIIβ interactions. A secondary consequence of destabilizing RIIβ dimers is the loss of intracellular targeting/anchoring capacity because monomers fail to bind AKAP75. Other NH₂-terminal residues directly modulate the affinity of RIIβ dimers for the AKAP75 tethering site. Replacement of Val²⁰-Leu²¹ with Ala-Ala produced a dimeric RIIβ protein that binds AKAP75 ∼4% as avidly as wild-type RIIβ. It is possible that the aliphatic side chains of Val²⁰ and Leu²¹ interact with the essential Leu and Ile residues in the AKAP75 tethering region.