RESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Apoptosis , Western Blotting , Proteínas Portadoras/genética , Caspasas/metabolismo , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Daño del ADN , Activación Enzimática , Glicoproteínas/genética , Células HEK293 , Células Hep G2 , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina/genética , Serina/metabolismo , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , ZixinaRESUMEN
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.