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J. Biol. Chem., Vol. 280, Issue 26, 25170-25177, July 1, 2005

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Cyclic AMP and AKAP-mediated Targeting of Protein Kinase A Regulates Lactate Dehydrogenase Subunit A mRNA Stability^*

Richard A. Jungmann and Olga Kiryukhina

From the Department of Cellular and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3008

Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies (Tian, D., Huang, D., Short, S., Short, M. L., and Jungmann, R. A. (1998) J. Biol. Chem. 273, 24861-24866) have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA subunits and AKAP 95 from RSW extracts by immunoprecipitation resulted in a marked loss of mRNA stabilization activity indicating that the presence of the PKA regulatory and catalytic subunits as well as AKAP 95 in the CSR-protein complexes was absolutely necessary to achieve LDH-A mRNA stabilization.

Received for publication, March 7, 2005 , and in revised form, May 4, 2005.

^* This work was supported by National Institutes of Health Grant GM53115. 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.

To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-0674; E-mail: rjungman{at}northwestern.edu.

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