Abstract
The 14-3-3-related protein SMG7 plays critical roles in regulation of DNA damage response and nonsense-mediated mRNA decay (NMD). Like 14-3-3, SMG7 engages phosphoserine-dependent protein interactions; however, the precise role of phosphorylation-mediated SMG7 binding remains unknown. Here, we show that DNA damage-induced SMG7-p53 binding requires phosphorylated Ser15 on p53, and that substitution of the conserved lysine residue K66 in the SMG7 14-3-3-like domain with the glutamic acid (E) abolishes interactions with its client proteins p53 and UPF1. Unexpectedly, loss of phosphoserine-dependent SMG7 binding does not significantly affect p53 stabilization/activation, and p53-dependent cell growth arrest or apoptosis upon DNA damage. Also surprisingly, cells expressing the SMG7 K66E-knockin mutant retain fully functional UPF1-mediated NMD. These findings are highly unusual, given that phosphorylation-mediated 14-3-3 binding has essential roles in numerous cellular signaling pathways. Thus, our studies suggest that 14-3-3-like proteins such as SMG7 likely function using additional distinct regulatory mechanisms besides phosphoserine-mediated protein interactions.
Highlights
The nonsense-mediated mRNA decay pathway (NMD) is a critical mRNA surveillance pathway, which degrades and eliminates aberrant mRNAs containing premature translation termination codons (PTC)[7]
As the 14-3-3-like domain of Suppressor for Morphological defects of Genitalia 7 (SMG7) is required for binding to UPF1 and may likely be involved in mediating DNA damage-induced interaction with p53, we investigated the role of SMG7 binding in the regulation of p53 and UPF1
In assessing the role of phosphorylation-mediated SMG7 binding to p53 and UPF1, we provide direct evidence to show a more complex SMG7 regulation of p53-mediated DNA damage response and UPF1-dependent NMD
Summary
The nonsense-mediated mRNA decay pathway (NMD) is a critical mRNA surveillance pathway, which degrades and eliminates aberrant mRNAs containing premature translation termination codons (PTC)[7]. Recent studies have identified Suppressor for Morphological defects of Genitalia 7 (SMG7) as a key factor in NMD and its N-terminal region is structurally homologous to 14-3-3 proteins[8,9] This 14-3-3-like domain of SMG7 resembles the 14-3-3 crescent shaped binding cleft and contains the critical conserved residue lysine 66 (K66) required for interaction with phosphorylated serine/threonine residues[8]. We identified SMG7 as a critical regulator for p53 stability and p53-mediated DNA damage response[19]. Beside regulating p53 stability, SMG7 functions via UPF1-mediated NMD to control the expression level of p53β, an alternatively spliced p53 isoform that is critically involved in cell cycle regulation and stress response[21,22,23,24,25]. In assessing the role of phosphorylation-mediated SMG7 binding to p53 and UPF1, we provide direct evidence to show a more complex SMG7 regulation of p53-mediated DNA damage response and UPF1-dependent NMD
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