Abstract
BackgroundOncoproteins encoded by the early region of adenoviruses have been shown to be powerful tools to study gene regulatory mechanisms, which affect major cellular events such as proliferation, differentiation, apoptosis and oncogenic transformation. They are possesing a key role to favor viral replication via their interaction with multiple cellular proteins. In a yeast two-hybrid screen we have identified Sprouty1 (Spry1) as a target of adenoviral E1A Oncoproteins. Spry proteins are central and complex regulators of the receptor tyrosine kinase (RTK) signalling pathway. The deregulation of Spry family members is often associated with alterations of the RTK signalling and its downstream effectors, leading to the ERK pathway.ResultsHere, we confirm our yeast two-hybrid data, showing the interaction between Spry1 and E1A in GST pull-down and immunoprecipitation assays. We also demonstrated the interaction of E1A with two further Spry isoforms. Using deletion mutants we identified the N-terminus and the CR conserved region (CR) 3 of E1A- and the C-terminal half of Spry1, which contains the highly conserved Spry domain, as the essential sites for direct interaction between Spry and E1A. Immunofluorescent microscopy data revealed a co-localization of E1A13S with Spry1 in the cytoplasm. SRE and TRE reporter assays demonstrated that co-expression of Spry1 with E1A13S abolishes the inhibitory function of Spry1 in RTK signalling, which is consequently accompanied with a decrease of E1A13S-induced gene expression.ConclusionsThese results establish Spry1 as a cytoplasmic localized cellular target for E1A oncoproteins to regulate the RTK signalling pathway, and consequently cellular events downstream of RTK that are essential for viral replication and transformation.
Highlights
Oncoproteins encoded by the early region of adenoviruses have been shown to be powerful tools to study gene regulatory mechanisms, which affect major cellular events such as proliferation, differentiation, apoptosis and oncogenic transformation
We surprisingly detected an interaction between Spry1 and the analog aminoterminal deletion mutant of the E1A13S isoform, indicating that the conserved region 3 (CR3) is involved in binding with Spry1
Our data showed that the C-terminal half of Spry1, bearing the highly conserved Spry domain, is necessary for the interaction with early transcription unit 1A (E1A), whereas the tyrosine-containing sequence of the N-terminal part is not essential for interaction (Figure 1C)
Summary
Oncoproteins encoded by the early region of adenoviruses have been shown to be powerful tools to study gene regulatory mechanisms, which affect major cellular events such as proliferation, differentiation, apoptosis and oncogenic transformation. They are possesing a key role to favor viral replication via their interaction with multiple cellular proteins. Proteins encoded by the early transcription unit 1A (E1A) of Adenovirus (Ad) are essential for the viral life cycle because of their necessity in regulating the expression of all other viral genes [1] In addition these proteins modulate the expression of specific cellular genes in infected cells to facilitate viral reproduction [2,3]. A few cytoplasmic localized interaction partners of E1A have been identified including the regulatory subunit II of protein kinase A (PKA-RIIa) [14], the receptor for activated C-kinase l (Rackl) [15,16], and the cytoplasmic proteasome 26S [17]
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