The RACK1 Signaling Scaffold Protein Selectively Interacts with the cAMP-specific Phosphodiesterase PDE4D5 Isoform

doi:10.1074/jbc.274.21.14909

The RACK1 Signaling Scaffold Protein Selectively Interacts with the cAMP-specific Phosphodiesterase PDE4D5 Isoform*

From the ^‡Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, United Kingdom and ^§Veterans Affairs Medical Center, Departments of Medicine (Hematology/Oncology) and Oncological Science, University of Utah, Salt Lake City, Utah 84148

Abstract

The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, β’-coatomer protein and G_sβ, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (K _a approximately 7 pm) when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3–4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.

Footnotes

↵* This work was supported in part by a Merit Review Award from the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to G. B. B.) and by a grant from the Medical Research Council (UK) (to M. D. H.).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.
↵¶ Supported by a collaborative Travel Grant from the Wellcome Trust, UK.
↵‖ To whom correspondence should be addressed: Veterans Affairs Medical Center, Depts. of Medicine (Hematology/Oncology) and Oncological Science, University of Utah, 500 Foothill Blvd., Salt Lake City, UT 84148. Tel.: 801-582-1565; Fax: 801-583-9624; E-mail:Graeme.Bolger{at}m.cc.utah.edu.
Abbreviations:

PDE(s)

cyclic nucleotide phosphodiesterase(s)

PDE4(s)

cAMP-specific phosphodiesterase(s)

β’-COP

β’-coatomer protein

GST

glutathione S-transferase

GST-RACK1

a fusion protein of GST and RACK1

MBP

maltose-binding protein

MBP-PDE4D3 and MBP-PDE4D5

fusion proteins of MBP and PDE4D3 or PDE4D5, respectively

ORF

open reading frame

PKC

protein kinase C

PCR

polymerase chain reaction

PMA

phorbol 2-myristate 3-acetate

RACK1

receptor for activated protein C kinase

rolipram

4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone

UCR

upstream conserved region

WD-repeat

a protein sequence motif containing repetitive sequences bounded by tryptophan and aspartic acid residues

VSV

vesicular stomatitis virus

ELISA

enzyme-linked immunosorbent assay

PAGE

polyacrylamide gel electrophoresis
- Received November 11, 1998.
- Revision received January 26, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.