Abstract
The structurally related ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases fulfill overlapping yet non-redundant functions as key regulators of cellular DNA damage responses. We recently showed that ATM phosphorylates the cyclic AMP response element-binding protein, CREB, following exposure to ionizing radiation (IR) and other DNA-damaging stimuli. Here, we show that a phospho-specific antibody recognizing the major ATM phosphorylation site in CREB cross-reacts with SV40 large tumor antigen (LTag), a multifunctional oncoprotein required for replication of the SV40 minichromosome. The relevant IR-induced phosphorylation site in LTag recognized by phospho-CREB antibody was mapped to Ser-120. IR strongly induced the phosphorylation of Ser-120 in an ATM-dependent manner in mouse embryo fibroblasts. Infection of African green monkey CV1 cells with SV40 resulted in the activation of ATM and phosphorylation of LTag and endogenous ATM substrates. Infection-induced LTag phosphorylation correlated with the onset of DNA replication, was ATM-dependent, and peaked when viral DNA levels reached their maximum. SV40 replication in CV1 cells required an intact LTag Ser-120 phosphorylation site and was inhibited following transfection with ATM small interfering RNA suggesting that ATM is required for optimal SV40 replication in primate cells. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.
Highlights
Display highly overlapping substrate specificities in vitro [1,2,3]
This experiment confirmed that ␣-cyclic AMP response element-binding protein (CREB)-pS121 showed strong reactivity with large tumor antigen (LTag) prepared from irradiated ATMϩ/ϩ cells, some residual reactivity was observed in the absence of ATM (Fig. 2A)
From these findings we conclude that ATM phosphorylates LTag in cellulo on one or more residues that are recognized by the ␣-CREB-pS121 antibody and that other kinases may contribute to the phosphorylation of this site
Summary
Display highly overlapping substrate specificities in vitro [1,2,3]. Substrates for ATM and ATR include the p53 and BRCA1 tumor suppressors among many other proteins involved in cell cycle checkpoint activation, DNA repair, and transcriptional regulation [1, 3]. This result corroborates the in vitro phosphorylation data and strongly implies that ATM phosphorylates LTag on Ser-120 in cellulo in response to DNA damage. A more extensive time course analysis revealed that ATM activation was not observed before 12 HPI and that both ATM autophosphorylation and LTag phosphorylation declined at late time points (48 – 60 h) after infection, when cells began to exhibit a viral cytopathic effect (CPE) (Fig. 3B).
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