Adeno-Associated Virus Rep78 Protein Interacts with Protein Kinase A and Its Homolog PRKX and Inhibits CREB-Dependent Transcriptional Activation

Abstract

Adeno-associated virus (AAV) is a human parvovirus of the genusDependovirus. AAV replication is largely restricted to cells which are coinfected with a helper virus. In the absence of a helper virus, the AAV genome can integrate into a specific chromosomal site where it remains latent until reactivated by superinfection of the host cell with an appropriate helper virus. Replication functions of AAV have been mapped to the Rep68 and Rep78 gene products. Rep proteins demonstrate DNA binding, endonuclease, and helicase activities and are involved in regulation of transcription from both AAV and heterologous promoters. AAV has been associated with suppression of oncogenicity in a range of viral and nonviral tumors. In this study we sought to identify and study cellular protein targets of AAV Rep, in order to develop a better understanding of the various activities of Rep. We used the yeast two-hybrid system to identify HeLa cell proteins that interact with AAV type 2 Rep78. We isolated several strongly interacting clones which were subsequently identified as PRKX (previously named PKX1), a recently described homolog of the protein kinase A (PKA) catalytic subunit (PKAc). The interaction was confirmed in vitro by using pMal-Rep pull-down assays. The region of Rep78 which interacts was mapped to a C-terminal zinc finger-like domain; Rep68, which lacks this domain, did not interact with PRKX. PRKX demonstrated autophosphorylation and kinase activity towards histone H1 and a PKA oligopeptide target. Autophosphorylation was inhibited by interaction with Rep78. In transfection assays, a PRKX expression vector was shown to be capable of activating CREB-dependent transcription. This activation was suppressed by Rep78 but not by Rep68. Since PRKX is a close homolog of PKAc, we investigated whether Rep78 could interact directly with PKAc. pMal-Rep78 was found to associate with purified PKAc and inhibited its kinase activity. Cotransfection experiments demonstrated that Rep78 could block the activation of CREB by a PKAc expression vector. These experiments suggest that AAV may perturb normal cyclic AMP response pathways in infected cells.

PRKX, TTBK2 and RSK4 expression causes sunitinib resistance in kidney carcinoma‐ and melanoma‐cell lines

“…PRKX is involved in renal development, regulating epithelial cell migration, uretic bud branching and induction of glomeruli formation, indicating its oncogenic potential. 23 Mechanistically, PRKX is capable to activate CREB-dependent transcription in vitro, 24 whereas the transcription factor CREB induces the expression of the Microphthalmia-associated transcription factor (MITF). 25 As our study revealed a correlation between an elevated PRKX expression and Sunitinib resistance formation, we postulate a role of PRKX in increasing MITF expression and/or activity.…”

Altered Biology of Adeno-associated Virus Type 2 and Human Papillomavirus during Dual Infection of Natural Host Tissue

“…Binding to Rep78 inhib- ited autophosphorylation and kinase activity of PRKX. PRKX also mediated CREB-dependent transcription, which was suppressed by Rep78 but not Rep68 (di Pasquale and Stacey, 1998). We have found that the presence or absence of protein kinase C activators greatly affects the ability of AAV to inhibit HPV replication (Alam and Meyers, unpublished observations).…”

“…Rep78 has been shown to disrupt binding of TBP to the HPV16 p97 promoter, suggesting an indirect effect of Rep78 on HPV promoter expression (Su et al, 2000). Recent yeast two-hybrid screens and in vitro studies suggest that Rep78 may interfere with normal cyclic AMP response pathways in AAV-infected cells (Chiorini et al, 1998;di Pasquale and Stacey, 1998). Two-hybrid studies have implicated that the Rep78 C-terminal domain binds to PRKX, a homolog of the protein kinase A (PKA) catalytic subunit (Chiorini et al, 1998;di Pasquale and Stacey, 1998), which was shown to undergo autophosphorylation and demonstrated kinase activity toward a PKA oligopeptide target (di Pasquale and Stacey, 1998).…”

PRKX, a Novel cAMP-Dependent Protein Kinase Member, Plays an Important Role in Development

“…Reduction of PRKX, TTBK2 or RSK4 triggers sunitinib sensitization and increases sunitinib inhibition of cancer cell migration [Bender and Ullrich, ]. Mechanistically, PRKX was shown to be capable of activating CREB‐dependent transcription in vitro [Di Pasquale and Stacey, ], and the transcription factor CREB induces expression of the Microphthalmia‐associated transcription factor (MITF) [Saha et al, ]. Therefore, PRKX expression increases MITF expression and enhances sunitinib resistance.…”