Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase IIβ to the cytoskeleton

S. B. Glantz, Y. Li, C. S. Rubin

Research output: Contribution to journalArticle

78 Scopus citations

Abstract

Cyclic AMP-dependent protein kinase IIβ (PKAIIβ) is the principal mediator of cAMP action in neurons. A Kinase Anchor Proteins (AKAPs) are enriched in forebrain neurons and have distinct high affinity binding domains for the regulatory subunit (RIIβ) of PKAIIβ and components of the dendritic cytoskeleton. The selective accumulation of AKAP · RIIβ complexes near dendritic microtubules tethers PKAIIβ in proximity with adenylate cyclase in the synaptic plasma membrane and cytoskeletal proteins that are substrates for the kinase, thereby creating intraneuronal target sites for signals carried by cAMP. We have characterized the targeting (anchoring) and tethering (RIIβ binding) domains of a prototypic anchor protein AKAP75. Deletion of N-terminal residues 27-48 generated a truncated RIIβ-binding protein that partitions equally between the cytosol and detergent-insoluble fractions of HEK293 cells. Further removal of a non-adjacent sequence (residues 77-91) produced a cytosolic protein with unimpaired RIIβ binding activity. Thus, two non-contiguous domains mediate the intracellular localization of AKAP75. Boundaries for the RIIβ tethering domain were mapped to residues 392-413 by scanning mutagenesis. Residues containing long aliphatic side chains are essential for the high affinity binding of RIIβ by AKAP75. Contributions of hydrophobic amino acids to tethering activity also depend on the position of the residue in the sequence. Certain conservative mutations that should not alter significantly the overall hydrophobicity or helicity of the tethering region (e.g. replacement of Leu with Ala) diminish the RIIβ binding activity of AKAP75.

Original languageEnglish (US)
Pages (from-to)12796-12804
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number17
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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