Mutagenesis of the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta reveals hydrophobic amino acids that are essential for RII beta dimerization and/or anchoring RII beta to the cytoskeleton.
In neurons cAMP-dependent protein kinase II beta (PKAII beta) is sequestered in the dendritic cytoskeleton because the regulatory subunit (RII beta) of the enzyme is tightly bound by A Kinase Anchor Proteins (AKAPs). The prototypic neuronal anchor protein AKAP75 has a COOH-terminal 22-residue RII beta 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 RII beta. Mutagenesis, recombinant protein expression, and physicochemical characterization were used to investigate the structural basis for the homodimerization and AKAP75 binding activities of RII beta. 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 RII beta subunits that cannot bind AKAP75. The results are not due to general misfolding since mutant RII beta monomers bind cAMP and inhibit the catalytic subunit of PKAII beta with the same affinity and efficacy as wild-type RII beta 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 RII beta-RII beta interactions. A secondary consequence of destabilizing RII beta dimers is the loss of intracellular targeting/anchoring capacity because monomers fail to bind AKAP75. Other NH2-terminal residues directly modulate the affinity of RII beta dimers for the AKAP75 tethering site. Replacement of Val20-Leu21 with Ala-Ala produced a dimeric RII beta protein that binds AKAP75 approximately 4% as avidly as wild-type RII beta. 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.
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Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
Rubin
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D017931:DNA Primers; D046911:Macromolecular Substances; C517766:PRKAR1B protein, human; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases