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Molecular and Cellular Biology, October 1998, p. 5921-5929, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Inhibition of PrKX, a Novel Protein Kinase, and the Cyclic AMP-Dependent Protein Kinase PKA by the Regulatory Proteins of Adeno-Associated Virus Type 2

John A. Chiorini,1 Bastian Zimmermann,2 Linda Yang,1 Richard H. Smith,1 Aaron Ahearn,1 Friedrich Herberg,2 and Robert M. Kotin1,*

Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892,1 and Institute for Physiological Chemistry, Ruhr-University Bochum, 44780 Bochum, Germany2

Received 9 January 1998/Returned for modification 10 March 1998/Accepted 16 July 1998

Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40. Expression of these nonstructural proteins affects cell growth and gene expression through processes that have not yet been characterized. Using a yeast two-hybrid screen, we have demonstrated that a stable interaction occurs between the viral proteins Rep78 and Rep52 and the putative protein kinase PrKX, which is encoded on the X chromosome. The stability and specificity of the Rep-PrKX interaction were confirmed by coimmunoprecipitation of complexes assembled in vitro and in vivo. Overexpressed PrKX, which was purified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inhibited by stoichiometric amounts of Rep52 and weakly inhibited with Rep68, which lacks the carboxy-terminal sequence contained in Rep52. Similarly, a stable interaction was observed with Rep78, which also contains the carboxy-terminal sequence of Rep52. A stable interaction and inhibition were also observed between Rep52 and the catalytic subunit of PKA. By using surface plasmon resonance and kinetic studies, Kis of approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX, respectively. Thus, Rep52 but not Rep68 can significantly inhibit the trans- and autophosphorylation activities of these kinases. The biological effects of Rep78-specific inhibition of PKA-responsive genes are illustrated by the reduction of steady-state levels of cyclic AMP-responsive-element-binding protein and cyclin A protein.

* Corresponding author. Mailing address: NIH/NHLBI/MHB, Bldg. 10/7D18, 10 Center Dr. MSC 1654, Bethesda, MD 20892-1654. Phone: (301) 496-1594. Fax: (301) 496-9985. E-mail: kotinr{at}fido.nhlbi.nih.gov.

Molecular and Cellular Biology, October 1998, p. 5921-5929, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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