Adeno-associated virus type 2 Rep78 inhibition of PKA and PRKX: Fine mapping and analysis of mechanism

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.