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Journal of Virology, February 2002, p. 1033-1042, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1033-1042.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Adeno-Associated Virus Type 2 Rep78 Inhibition of PKA and PRKX: Fine Mapping and Analysis of Mechanism

Michael Schmidt, John A. Chiorini,,{dagger} Sandra Afione, and Robert Kotin*

Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Received 3 August 2001/ Accepted 26 October 2001

Hormones and neurotransmitters utilize cyclic AMP (cAMP) as a second messenger in signal transduction pathways to regulate cell growth and division, differentiation, gene expression, and metabolism. Adeno-associated virus type 2 (AAV-2) nonstructural protein Rep78 inhibits members of the cAMP signal transduction pathway, the protein kinases PKA and PRKX. We mapped the kinase binding and inhibition domain of Rep78 for PRKX to amino acids (aa) 526 to 561 and that for PKA to aa 526 to 621. These polypeptides were as potent as full-length Rep78 in kinase inhibition, which suggests that the kinase-inhibitory domain is entirely contained in these Rep peptides. Steady-state kinetic analysis of Rep78-mediated inhibition of PKA and PRKX showed that Rep78 appears to increase the Km value of the peptide kinase substrate, while the maximal velocity of the reaction was unaffected. This indicates that Rep78 acts as a competitive inhibitor with respect to the peptide kinase substrate. We detected homology between a cellular pseudosubstrate inhibitor of PKA, the protein kinase inhibitor PKI, and the PRKX and PKA inhibition domains of Rep78. Due to this homology and the competitive inhibition mechanism of Rep78, we propose that Rep78 inhibits PKA and PRKX kinase activity by pseudosubstrate inhibition.

* Corresponding author. Mailing address: LBG, NHLBI, Bldg. 10, Rm. 7D05, Bethesda, MD 20892-1654. Phone: (301) 496-1594. Fax: (301) 496-9985. E-mail: kotinr{at}nhlbi.nih.gov.

{dagger} Present address: Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892.

Journal of Virology, February 2002, p. 1033-1042, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1033-1042.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Nash, K., Chen, W., Salganik, M., Muzyczka, N. (2009). Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein. J. Virol. 83: 454-469 [Abstract] [Full Text]  
  • Bao, X., Kolli, D., Liu, T., Shan, Y., Garofalo, R. P., Casola, A. (2008). Human Metapneumovirus Small Hydrophobic Protein Inhibits NF-{kappa}B Transcriptional Activity. J. Virol. 82: 8224-8229 [Abstract] [Full Text]  
  • Li, W., Yu, Z.-X., Kotin, R. M. (2005). Profiles of PrKX Expression in Developmental Mouse Embryo and Human Tissues. J. Histochem. Cytochem. 53: 1003-1009 [Abstract] [Full Text]  


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