Ubiquitination and Degradation of Homeodomain-interacting Protein Kinase 2 by WD40 Repeat/SOCS Box Protein WSB-1

Abstract

Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53-and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses.

MYCN Sensitizes Human Neuroblastoma to Apoptosis by HIPK2 Activation through a DNA Damage Response

“…In particular, we showed that this proapoptotic phenotype of MYCN-overexpressing cells also required p53 phosphorylation at serine 46 and were associated to a more pronounced activation of proapoptotic target genes compared with those involved in cell cycle arrest, such as the p21 WAF1 , in keeping with the current understanding of p53 activity (32,33). Among a few kinases able to phosphorylate p53 S46 , we focused our attention on HIPK2, a protein required for apoptosis induced by neurotrophin deprivation in developing sympathetic neurons (45), whose role in p53 S46 phosphorylation and p53-dependent apoptosis has clearly been established (26,36 (37)(38)(39)(40). DNA damage was shown to promote HIPK2 accumulation through an ATM/ATRdependent inhibition of its degradation by Siah1 and/or WSB1 ubiquitin ligases (38,40).…”

“…Among a few kinases able to phosphorylate p53 S46 , we focused our attention on HIPK2, a protein required for apoptosis induced by neurotrophin deprivation in developing sympathetic neurons (45), whose role in p53 S46 phosphorylation and p53-dependent apoptosis has clearly been established (26,36 (37)(38)(39)(40). DNA damage was shown to promote HIPK2 accumulation through an ATM/ATRdependent inhibition of its degradation by Siah1 and/or WSB1 ubiquitin ligases (38,40). Oncogene-dependent replication stress was also shown to induce DNA damage and ATM/ATR activation leading to p53-dependent apoptosis (or senescence), suggesting that DDR might represent an endogenous anticancer barrier contrasting transforma- tion induced by several oncogenes (41).…”

“…3D), suggesting that HIPK2 induction by MYCN might occur at the level of protein stability. HIPK2 expression is largely controlled via proteasomal degradation by several E3 ubiquitin ligases (37)(38)(39)(40). Importantly, in response to DNA damage, the ATM and ATR kinases were shown to promote HIPK2 accumulation by inhibiting its ubiquitin ligases Siah1 and WSB1 (38,40).…”

“…HIPK2 expression is largely controlled via proteasomal degradation by several E3 ubiquitin ligases (37)(38)(39)(40). Importantly, in response to DNA damage, the ATM and ATR kinases were shown to promote HIPK2 accumulation by inhibiting its ubiquitin ligases Siah1 and WSB1 (38,40). By inducing replication stress, several oncogenes were shown to initiate a DDR sustained by the activation of the ATM/ATR kinases (41); thus, we postulated that MYCN might promote HIPK2 accumulation as a consequence of DDR.…”

Regulation of p53 activity by HIPK2: molecular mechanisms and therapeutical implications in human cancer cells

“…HIPK2 downregulation during hypoxia strongly reduces ADR-induced p53Ser46 phosphorylation, moreover, it allows the HIPK2 elimination from promoter-associated repressor complexes with induction of a substantial fraction of hypoxia-induced genes such as PDK1, TGFB3, CYP1B1 (cytochrome P450) (Calzado et al, 2009a) and hypoxia-induced factor (HIF)-1a (Nardinocchi et al, 2009). Other ubiquitin ligases involved in HIPK2 downregulation may take place during hypoxia including WD40-repeat/SOCS box protein WSB-1 (Choi et al, 2008), which is a novel predicted HIF1 target gene, as identified by an integrative genomic approach, upregulated upon hypoxia (Benita et al, 2009); and the RING family member Siah-1, originally identified along with Siah-2 as HIPK2-interacting protein (Winter et al, 2009;Calzado et al, 2009a). As for WSB-1 and Siah-2, Siah-1 depletion by siRNA partially inhibits hypoxia-induced HIPK2 degradation (Moehlenbrink et al, 2010).…”