Cloning and Characterization of A-kinase Anchor Protein 100 (AKAP100)

doi:10.1074/jbc.270.16.9327

Cloning and Characterization of A-kinase Anchor Protein 100 (AKAP100)

A PROTEIN THAT TARGETS A-KINASE TO THE SARCOPLASMIC RETICULUM (*)

From the (1) Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

^§ To whom correspondence should be addressed:
Vollum Institute L-474, Oregon Health Sciences University, Portland, OR 92701-3098.
Tel.: 503-494-4652; Fax: 503-494-2285.

Abstract

Differential localization of the type II cAMP-dependent protein kinase (PKA) is achieved by interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Anchoring is a likely means to adapt PKA for regulation of cAMP-responsive events through colocalization of the kinase with preferred substrates. Using an interaction cloning strategy with an RIIα protein probe, we have identified a 655-amino acid protein (named AKAP100). Recombinant AKAP100, expressed in Escherichia coli, binds RIIα in a solid-phase overlay assay. The cellular and subcellular distribution of AKAP100 was analyzed by various methods. Northern blot analysis with the AKAP100 cDNA as a probe detected an 8-kilobase message in some human tissues including various brain regions; however, the message was predominately expressed in cardiac and skeletal muscle. Anti-AKAP100 antibodies confirmed expression in the rat cardiac and skeletal muscle cell lines, H9c2 and L6P, whereas immunohistochemical analysis revealed that AKAP100 was localized to the sarcoplasmic reticulum of both cell types. RII was also detected in these regions. AKAP100 was detected in preparations of RII purified from L6P cell extracts by cAMP-agarose affinity chromatography. Collectively, these results suggest that AKAP100 functions to maintain the type II PKA at the sarcoplasmic reticulum.

Footnotes

↵* This work was supported by National Institutes of Health Grant GM 48231 (to J. D. S.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U17195.
↵¹ The abbreviations used are:

PKA

cAMP-dependent protein kinase

AKAP

A-kinase anchor protein

bp

base pair(s)

PBS

phosphate-buffered saline

BSA

bovine serum albumin

PAGE

polyacrylamide gel electrophoresis.
↵²B. M. Little and J. D. Scott, unpublished observation.