TYROSINE KINASE-DEPENDENT RELEASE OF AN ADENOVIRUS PRETERMINAL PROTEIN COMPLEX FROM THE NUCLEAR MATRIX

Adenovirus (Ad) replicative complexes form at discrete sites on the nu clear matrix (NM) through the interaction of Ad preterminal protein (p TP). The NM is a highly salt-resistant fibrillar network which is know n to anchor transcription, mRNA splicing, and DNA replication complexe s. Incubation of rATP with NM to which pTP was bound caused the releas e of pTP as a pTP-NM complex with a size of 220 to 230 kDa; incubation with 5' adenylylimidodiphosphate (rAMP-PNP) showed no significant rel ease, indicating that rATP hydrolysis was required. With NM extracts, it was shown that a pTP NM complex which was capable of binding Ad ori gin DNA could be reconstituted in vitro. A number of high molecular-we ight NM proteins ranging in size from 120 to 200 kDa were identified o n Far Western blots for their ability to bind pTP. rATP-dependent rele ase of pTP from the NM was inhibited in a dose-dependent fashion by th e addition of tyrosine kinase inhibitors, such as quercetin, methyl-2, 5-dihydroxycinnamate, or genistein. Nhl-mediated phosphorylation of a poly(Glu, Tyr) substrate was also significantly abrogated by the addit ion of these compounds, rATP-dependent release of Ad ]DNA termini boun d to the NM via pTP was also blocked by the addition of these inhibito rs. These results indicate that a tyrosine kinase mechanism controls t he release of pTP from its binding sites on the NM. These data support the concept that phosphorylation may play a key role in the modulatio n of pTP binding sites on the NM.