INHIBITION OF PRKX, A NOVEL PROTEIN-KINASE, AND THE CYCLIC-AMP-DEPENDENT PROTEIN-KINASE PKA BY THE REGULATORY PROTEINS OF ADENOASSOCIATED VIRUS TYPE-2

Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40, Expression of these nonstruct ural proteins affects cell growth and gene expression through processe s that have not yet been characterized. Using a yeast two-hybrid scree n, we have demonstrated that a stable interaction occurs between the v iral proteins Rep78 and Rep52 and the putative protein kinase PrKX, wh ich is encoded on the X chromosome. The stability and specificity of t he Rep-PrKX interaction were confirmed by coimmunoprecipitation of com plexes assembled in vitro and in vivo. Overexpressed PrKX, which was p urified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inh ibited by stoichiometric amounts of Rep52 and weakly inhibited with Re p68, which lacks the carboxy-terminal sequence contained in Rep52, Sim ilarly, a stable interaction was observed with Rep78, which also conta ins the carboxy-terminal sequence of Rep52. A stable interaction and i nhibition were also observed between Rep52 and the catalytic subunit o f PKA, By using surface plasmon resonance and kinetic studies, K(i)s o f approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX, respectively. Thus, Rep52 but not Rep68 can significantly i nhibit 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 cycl ic AMP-responsive-element-binding protein and cyclin A protein.