title: Adeno-associated virus type 2 Rep78 inhibition of PKA and PRKX: fine mapping and analysis of mechanism.
abstract: 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 K(m) 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.
authors.affiliation: Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Schmidt
authors.forename: John A
authors.initials: JA
authors.lastname: Chiorini
authors.forename: Sandra
authors.initials: S
authors.lastname: Afione
authors.forename: Robert
authors.initials: R
authors.lastname: Kotin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D002874:Chromosome Mapping; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D014764:Viral Proteins; C015482:protein kinase modulator; C068734:rep proteins, Adeno-associated virus 2; C109450:PRKX protein, human; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases