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J. Biol. Chem., Vol. 279, Issue 8, 7029-7036, February 20, 2004

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cAMP-dependent Protein Kinase Regulatory Subunit Type II

ACTIVE SITE MUTATIONS DEFINE AN ISOFORM-SPECIFIC NETWORK FOR ALLOSTERIC SIGNALING BY cAMP^*

Kerri M. Zawadzki and Susan S. Taylor¶||

From the Department of Chemistry and Biochemistry and the ^¶Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0654

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RII and two mutant RII subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The K_a for RII is 610 nM, which is 10-fold greater than its K_d(cAMP) and significantly higher than for RI and RII. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The K_a of the A domain mutant protein is 21-fold greater than that of wild-type and the K_d(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant K_a is 2-fold less than its K_d(cAMP), demonstrating that, unlike RI, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a K_a identical to the K_d(cAMP) of full-length RII, indicating that the B domain inhibits holoenzyme activation for RII. In RI, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.

Received for publication, October 1, 2003

^* This work was supported in part by National Institutes of Health (NIH) Grant GM34921 (to S. S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship and NIH Training Grant GM08326-10.

^|| To whom correspondence should be addressed. Tel.: 858-534-5554; Fax: 858-534-8193; E-mail: staylor{at}ucsd.edu.

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