The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells.
E2F transcription factors are implicated in diverse cellular functions. The founding member, E2F-1, is endowed with contradictory activities, being able to promote cell-cycle progression and induce apoptosis. However, the mechanisms that underlie the opposing outcomes of E2F-1 activation remain largely unknown. We show here that E2F-1 is directly methylated by PRMT5 (protein arginine methyltransferase 5), and that arginine methylation is responsible for regulating its biochemical and functional properties, which impacts on E2F-1-dependent growth control. Thus, depleting PRMT5 causes increased E2F-1 protein levels, which coincides with decreased growth rate and associated apoptosis. Arginine methylation influences E2F-1 protein stability, and the enhanced transcription of a variety of downstream target genes reflects increased E2F-1 DNA-binding activity. Importantly, E2F-1 is methylated in tumour cells, and a reduced level of methylation is evident under DNA damage conditions that allow E2F-1 stabilization and give rise to apoptosis. Significantly, in a subgroup of colorectal cancer, high levels of PRMT5 frequently coincide with low levels of E2F-1 and reflect a poor clinical outcome. Our results establish that arginine methylation regulates the biological activity of E2F-1 activity, and raise the possibility that arginine methylation contributes to tumourigenesis by influencing the E2F pathway.
Summary The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase (PRMT) 1 and symmetric dimethylating PRMT5, and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favours proliferation by antagonising methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN down-regulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.
HIPK2 activates the apoptotic arm of the DNA damage response by phosphorylating tumor suppressor p53 at serine 46. Unstressed cells keep HIPK2 levels low through targeted polyubiquitination and subsequent proteasomal degradation. Here we identify the LIM domain protein Zyxin as a novel regulator of the HIPK2-p53 signaling axis in response to DNA damage. Remarkably, depletion of endogenous Zyxin, which colocalizes with HIPK2 at the cytoskeleton and in the cell nucleus, stimulates proteasome-dependent HIPK2 degradation. In contrast, ectopic expression of Zyxin stabilizes HIPK2, even upon enforced expression of its ubiquitin ligase Siah-1. Consistently, Zyxin physically interacts with Siah-1, and knock-down of Siah-1 rescues HIPK2 expression in Zyxin-depleted cancer cells. Mechanistically, our data suggest that Zyxin regulates Siah-1 activity through interference with Siah-1 dimerization. Furthermore, we show that endogenous Zyxin coaccumulates with HIPK2 in response to DNA damage in cancer cells, and that depletion of endogenous Zyxin results in reduced HIPK2 protein levels and compromises DNA damage-induced p53 Ser46 phosphorylation and caspase activation. These findings suggest an unforeseen role for Zyxin in DNA damage-induced cell fate control through modulating the HIPK2-p53 signaling axis. Cancer Res; 71(6); 2350-9. Ó2011 AACR.
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