Mutagenesis of the Regulatory Subunit (RII) of cAMP-dependent Protein Kinase II Reveals Hydrophobic Amino Acids That Are Essential for RII Dimerization and/or Anchoring RII to the Cytoskeleton

doi:10.1074/jbc.270.4.1935

Mutagenesis of the Regulatory Subunit (RII) of cAMP-dependent Protein Kinase II Reveals Hydrophobic Amino Acids That Are Essential for RII Dimerization and/or Anchoring RII to the Cytoskeleton (*)

Ying Li and
Charles S. Rubin(§)

From the Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461

^§ To whom correspondence should be sent:
Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.
Tel.: 718-430-2505; Fax: 718-829-8705.

Abstract

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 Leu or Phe 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 Leu, Val, Leu, Phe, Leu, or Leu and replacement of Leu with Ile or Val did not impair the dimerization reaction. Evidently, large hydrophobic side chains of Leu and Phe 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 Val-Leu 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 Val and Leu interact with the essential Leu and Ile residues in the AKAP75 tethering region.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant GM22792. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKAIIβ

the type IIβ isoform of cyclic AMP-dependent protein kinase

PKA

cyclic AMP-dependent protein kinase

AKAP

protein kinase A anchor protein

R

regulatory subunit of cAMP-dependent protein kinase

RIIβ-N158

a protein with an amino acid sequence that corresponds to residues 1-158 at the NH₂ terminus of RIIβ

Mops

4-morpholinepropanesulfonic acid.
↵²Y. Li and C. S. Rubin, unpublished observation.
- Received August 29, 1994.
- Revision received November 11, 1994.