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

Susan B. Glantz, Ying Li, Charles S. Rubin

Research output: Contribution to journalArticle

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Abstract

Cyclic AMP-dependent protein kinase IIβ (PKAIIβ) is the principal mediator of cAMP action in neurons. A + (Combining low line sign) K + (Combining low line sign)inase A + (Combining low line sign)nchor P + (Combining low line sign)roteins (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 - Jun 15 1993

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Cyclic AMP-Dependent Protein Kinases
Cytoskeleton
Protein Kinases
Neurons
Microtubule Proteins
Mutagenesis
Cytoskeletal Proteins
Cell membranes
Hydrophobicity
Anchors
Adenylyl Cyclases
Detergents
Synaptic Membranes
Carrier Proteins
Membrane Proteins
Proteins
Phosphotransferases
HEK293 Cells
Prosencephalon
Scanning

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase IIβ to the cytoskeleton. / Glantz, Susan B.; Li, Ying; Rubin, Charles S.

In: Journal of Biological Chemistry, Vol. 268, No. 17, 15.06.1993, p. 12796-12804.

Research output: Contribution to journalArticle

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