A Kinase Anchor Protein 75 Targets Regulatory (RII) Subunits of cAMP-dependent Protein Kinase II to the Cortical Actin Cytoskeleton in Non-neuronal Cells

doi:10.1074/jbc.271.28.16862

A Kinase Anchor Protein 75 Targets Regulatory (RII) Subunits of cAMP-dependent Protein Kinase II to the Cortical Actin Cytoskeleton in Non-neuronal Cells*

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

^§ To whom correspondence should be addressed:
Dept. of Molecular Pharmacology, F-229, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.
Tel: 718-430-2505; Fax: 718-430-8922; E-mail: rubin{at}aecom.yu.edu.

Abstract

Neuronal A kinase anchor protein (AKAP) homologs, such as AKAPs 75 and 150, tether cAMP-dependent protein kinase II (PKAII) isoforms to the postsynaptic cytoskeleton, thereby creating target sites for cAMP action. These AKAPs, which bind regulatory subunits (RIIs) of PKAII, are also expressed in certain non-neuronal cells. Non-neuronal cell lines that stably express wild type and mutant AKAP75 transgenes were generated to investigate the extraneuronal function of AKAPs. In non-neuronal cells, AKAP75 accumulates selectively in the actin-rich, cortical cytoskeleton in close proximity with the plasma membrane. AKAP75 efficiently sequesters cytoplasmic RIIα and RIIβ (PKAII isoforms) and translocates these polypeptides to the cell cortex. Two structural modules in AKAP75, T₁ (residues 27-48), and T₂ (residues 77-100), are essential for targeting AKAP75·RII complexes to the cortical cytoskeleton. Deletions or amino acid substitutions in T₁ and/or T₂ result in the dispersion of both AKAP75 and RII subunits throughout the cytoplasm. AKAP75 is co-localized with F-actin and fodrin in the cortical cytoskeleton. Incubation of cells with 5 μM cytochalasin D disrupts actin filaments and dissociates actin from the cell cortex. In contrast, the bulk of AKAP75 and fodrin remain associated with the cortical region of cytochalasin D-treated cells. Thus, targeting of AKAP75 does not depend upon direct binding with F-actin. Rather, AKAP75 (like fodrin) may be associated with a multiprotein complex that interacts with integral plasma membrane proteins.

Footnotes

↵^‡ These two authors contributed equally to these studies.
↵* This work was supported in part by National Institutes of Health Grant GM22792 (to C. S. R.) and Medical Scientist Training Program Grant GM7288 (to C. N.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKA

protein kinase A (cAMP-dependent protein kinase)

RI and RII

regulatory subunits of protein kinase A I and II, respectively

AKAP

A kinase anchor protein

S-AKAP

spermatid AKAP

T₁ and T₂

targeting domains corresponding to residues 27-48 and 77-100 in AKAP75, respectively

PBS

phosphate-buffered saline.
↵² F. Dong, and C. S. Rubin, unpublished observations.
↵³ Y. Li, S. B. Glantz, and C. S. Rubin, unpublished observations.
↵⁴ The intracellular distribution of endogenous AKAP79 is similar to that of AKAP75. The low abundance AKAP79 polypeptide yields weak fluorescence signals that make a negligible contribution to the results reported in this paper.
- Received January 17, 1996.
- Revision received March 22, 1996.