Functional impairment of the HIPK2 small ubiquitin-like modifier (SUMO)-interacting motif in acute myeloid leukemia.

Covalent conjugations of the SUMO-1 moiety on a target protein play important roles in the regulation of cellular protein function. SUMO-conjugation of PML is a regulatory step for PML nuclear body (PML-NB) formation, and HIPK2 is SUMO-conjugated and recruited into the PML-NBs. Although HIPK2 mutations (R861W and N951I) were found in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients, little is known about the underlying mechanisms by which HIPK2 mutations are associated with the pathogenesis of leukemia. Here we show that HIPK2 mutants found in AML and MDS patients are defective in SUMO-interacting motif (SIM) function. Due to defective SIM function, the HIPK2 mutants were not modified with SUMO-1, and not recruited to the PML-NBs. However, the HIPK2 mutants can normally bind to and phosphorylate AML1b. Therefore, the HIPK2 mutants can sequestrate the AML1 complex out of the PML-NBs, resulting in the disruption of AML1-mediated activation of target genes for myeloid differentiation. In addition, the differentiation of K562 blast cells was impaired by the expression of the HIPK2 SIM-defective mutants. These results suggest that HIPK2 targeting into the PML-NBs via the SIMs is crucial for HIPK2-mediated induction of myeloid differentiation, and is associated with AML pathogenesis.

Glioma-derived cancer stem cells are hypersensitive to proteasomal inhibition.

Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem cells derived from gliomas, GSCs, are up to 1,000-fold more sensitive to PIs (IC50, 27-70 nM) compared with their differentiated controls (IC50, 47 to ¼100 µM). The stemness of GSCs correlates to increased ubiquitination, whose misregulation readily triggers apoptosis. PI-induced apoptosis of GSCs is independent of NF-κB but involves the phosphorylation of c-Jun N-terminal kinase as well as the transcriptional activation of endoplasmic reticulum (ER) stress-associated proapoptotic mediators. In contrast to the general notion that ER stress-associated apoptosis is signaled by prolonged unfolded protein response (UPR), GSC-selective apoptosis is instead counteracted by the UPR ATF3 is a key mediator in GSC-selective apoptosis. Pharmaceutical uncoupling of the UPR from its downstream apoptosis sensitizes GSCs to PIs in vitro and during tumorigenesis in mice. Thus, a combinational treatment of a PI with an inhibitor of UPR-coupled apoptosis may enhance targeting of stem cells in gliomas.

Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/GRP78/BiP.

The N-end rule pathway is a proteolytic system, in which single N-terminal residues act as a determinant of a class of degrons, called N-degrons. In the ubiquitin (Ub)-proteasome system, specific recognition components, called N-recognins, recognize N-degrons and accelerate polyubiquitination and proteasomal degradation of the substrates. In this study, we show that the pathway regulates the activity of the macroautophagic receptor SQSTM1/p62 (sequestosome 1) through N-terminal arginylation (Nt-arginylation) of endoplasmic reticulum (ER)-residing molecular chaperones, including HSPA5/GRP78/BiP, CALR (calreticulin), and PDI (protein disulfide isomerase). The arginylation is co-induced with macroautophagy (hereafter autophagy) as part of innate immunity to cytosolic DNA and when misfolded proteins accumulate under proteasomal inhibition. Following cytosolic relocalization and arginylation, Nt-arginylated HSPA5 (R-HSPA5) is targeted to autophagosomes and degraded by lysosomal hydrolases through the interaction of its N-terminal Arg (Nt-Arg) with ZZ domain of SQSTM1. Upon binding to Nt-Arg, SQSTM1 undergoes a conformational change, which promotes SQSTM1 self-polymerization and interaction with LC3, leading to SQSTM1 targeting to autophagosomes. Cargoes of R-HSPA5 include cytosolic misfolded proteins destined to be degraded through autophagy. Here, we discuss the mechanisms by which the N-end rule pathway regulates SQSTM1-dependent selective autophagy.

TRAIL-Induced Caspase Activation Is a Prerequisite for Activation of the Endoplasmic Reticulum Stress-Induced Signal Transduction Pathways.

It is well known that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be initially triggered by surface death receptors (the extrinsic pathway) and subsequently amplified through mitochondrial dysfunction (the intrinsic pathway). However, little is known about signaling pathways activated by the TRAIL-induced endoplasmic reticulum (ER) stress response. In this study, we report that TRAIL-induced apoptosis is associated with the endoplasmic reticulum (ER) stress response. Human colorectal carcinoma HCT116 cells were treated with TRAIL and the ER stress-induced signal transduction pathway was investigated. During TRAIL treatment, expression of ER stress marker genes, in particular the BiP (binding immunoglobulin protein) gene, was increased and activation of the PERK (PKR-like ER kinase)-eIF2α (eukaryotic initiation factor 2α)-ATF4 (activating transcription factor 4)-CHOP (CCAAT-enhancer-binding protein homologous protein) apoptotic signal transduction pathway occurred. Experimental data from use of a siRNA (small interfering RNA) technique, caspase inhibitor, and caspase-3-deficient cell line revealed that TRAIL-induced caspase activation is a prerequisite for the TRAIL-induced ER stress response. TRAIL-induced ER stress was triggered by caspase-8-mediated cleavage of BAP31 (B cell receptor-associated protein 31). The involvement of the proapoptotic PERK-CHOP pathway in TRAIL-induced apoptosis was verified by using a PERK knockout (PERK(-/-)) mouse embryo fibroblast (MEF) cell line and a CHOP(-/-) MEF cell line. These results suggest that TRAIL-induced the activation of ER stress response plays a role in TRAIL-induced apoptotic death.

Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding.

We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.

HSP90 inhibitor NVP-AUY922 enhances TRAIL-induced apoptosis by suppressing the JAK2-STAT3-Mcl-1 signal transduction pathway in colorectal cancer cells.

TRAIL has been shown to induce apoptosis in cancer cells, but in some cases, certain cancer cells are resistant to this ligand. In this study, we explored the ability of representative HSP90 (heat shock protein 90) inhibitor NVP-AUY922 to overcome TRAIL resistance by increasing apoptosis in colorectal cancer (CRC) cells. The combination of TRAIL and NVP-AUY922 induced synergistic cytotoxicity and apoptosis, which was mediated through an increase in caspase activation. The treatment of NVP-AUY922 dephosphorylated JAK2 and STAT3 and decreased Mcl-1, which resulted in facilitating cytochrome c release. NVP-AUY922-mediated inhibition of JAK2/STAT3 signaling and down-regulation of their target gene, Mcl-1, occurred in a dose and time-dependent manner. Knock down of Mcl-1, STAT3 inhibitor or JAK2 inhibitor synergistically enhanced TRAIL-induced apoptosis. Taken together, our results suggest the involvement of the JAK2-STAT3-Mcl-1 signal transduction pathway in response to NVP-AUY922 treatment, which may play a key role in NVP-AUY922-mediated sensitization to TRAIL. By contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We provide a clinical rationale that combining HSP90 inhibitor with TRAIL enhances therapeutic efficacy without increasing normal tissue toxicity in CRC patients.

Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA).

Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy.

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.

Mdm2 associates with Ras effector NORE1 to induce the degradation of oncoprotein HIPK1.

The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2-NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.

Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53.

Homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of various transcription factors including p53 and CtBP in the DNA damage signaling pathway. PML-nuclear body (NB) is required for HIPK2-mediated p53 phosphorylation at Ser46 and induction of apoptosis. Although PML-NB targeting of HIPK2 has been shown, much is not clear about the molecular mechanism of HIPK2 recruitment to PML-NBs. Here we show that HIPK2 colocalizes specifically with PML-I and PML-IV. Mutational analysis showed that HIPK2 recruitment to PML-IV-NBs is mediated by the SUMO-interaction motifs (SIMs) of both PML-IV and HIPK2. Wild-type HIPK2 associated with SUMO-conjugated PML-IV at a higher affinity than with un-conjugated PML-IV, while the association of a HIPK2 SIM mutant with SUMO-modified PML-IV was impaired. In colony formation assays, HIPK2 strongly suppressed cell proliferation, but HIPK2 SIM mutants did not. In addition, activation and phosphorylation of p53 at the Ser46 residue were impaired by HIPK2 SIM mutants. These results suggest that SIM-mediated HIPK2 targeting to PML-NBs is crucial for HIPK2-mediated p53 activation and induction of apoptosis.

Pellino-1, an adaptor protein of interleukin-1 receptor/toll-like receptor signaling, is sumoylated by Ubc9.

Covalent modifications of the Pellino-1 protein are essential for transmitting innate immune response signals downstream, as the phosphorylation and polyubiquitination of Pellino-1 mediated by the IRAK proteins appear to have roles in regulating Pellino-1 function. In this study, we demonstrate that the Pellino-1 protein is post-translationally modified by small-ubiquitin-related modifier-1 (SUMO-1). Sumoylation assays with Pellino-1 and SUMO-1 expression plasmids reveal that the Pellino-1 protein is sumoylated in vitro and in vivo. Treatment of SUMO-1 specific protease 1 (SENP1) inhibited the sumoylation of the Pellino-1 protein and a GST pull-down assay as well as a yeast two hybrid assay showed that Pellino-1 binds to the SUMO-conjugating enzyme, Ubc9. Furthermore, we identified the five lysine residues of the Pellino-1 protein where SUMO-1 covalently attaches. Some of the sumoylated sites overlap with previously identified ubiquitination sites, suggesting competition between sumoylation and ubiquitination, as well as suggesting that the sumoylated Pellino-1 protein may have a cellular function distinct from previously identified functions.

Homeodomain-interacting protein kinase 2 (HIPK2) targets beta-catenin for phosphorylation and proteasomal degradation.

The regulation of intracellular beta-catenin levels is central in the Wnt/beta-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/beta-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of beta-catenin and results in the accumulation of beta-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of beta-catenin. HIPK2 phosphorylates beta-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of beta-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of beta-catenin, thereby potentiated beta-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of beta-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of beta-catenin through its phosphorylation and proteasomal degradation.

Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification.

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are approximately 96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralog-selective modification.

Ubiquitination and degradation of homeodomain-interacting protein kinase 2 by WD40 repeat/SOCS box protein WSB-1.

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.

12(S)-HPETE induces itch-associated scratchings in mice.

The itch-associated responses evoked by intradermal injection of 12(S)-HPETE and leukotriene B4 were compared in ICR-mice. 12(S)-HPETE and leukotriene B4 (0.01-0.2 nmol/site) induced scratching of the injected site, respectively; the dose-responses were a peak at 0.05 nmol/site (12(S)-HPETE) or 0.03 nmol/site (leukotriene B4). The scratching response by 12(S)-HPETE (0.05 nmol/site) started within 1 min, peaked in the first 10 min period, had almost subsided by 25 min whereas the effect of leukotriene B4 peaked in the second 10 min. The effect of leukotriene B4 is slightly stronger than that of 12(S)-HPETE in 40 min of count. The scratching induced by 12(S)-HPETE was inhibited by capsaicin, naltrexon, and LY255283. These results suggest the possibility that 12-lipoxygenase product can be added to a new member of an endogenous itch mediator in the skin.

Desumoylation of homeodomain-interacting protein kinase 2 (HIPK2) through the cytoplasmic-nuclear shuttling of the SUMO-specific protease SENP1.

The modification of homeodomain-interacting protein kinase 2 (HIPK2) by small ubiquitin-like modifier 1 (SUMO-1) plays an important role in its targeting into the promyelocytic leukemia body, as well as in its differential interaction with binding partner, but the desumoylation of HIPK2 by SUMO-specific proteases is largely unknown. In this study, we show that HIPK2 is a desumoylation target for the SUMO-specific protease SENP1 that shuttles between the cytoplasm and the nucleus. Mutation analyses reveal that SENP1 contains the nuclear export sequence (NES) within the extreme carboxyl-terminal region, and SENP1 is exported to the cytoplasm in a NES-dependent manner. Sumoylated HIPK2 are deconjugated by SENP1 both in vitro and in cultured cells, and the desumoylation is enhanced either by the forced translocation of SENP1 into the nucleus or by the SENP1 NES mutant. Concomitantly, desumoylation induces dissociation of HIPK2 from nuclear bodies. These results demonstrate that HIPK2 is a target for SENP1 desumoylation, and suggest that the desumoylation of HIPK2 may be regulated by the cytoplasmic-nuclear shuttling of SENP1.

Differential interactions of the homeodomain-interacting protein kinase 2 (HIPK2) by phosphorylation-dependent sumoylation.

Homeodomain-interacting protein kinase 2 (HIPK2) interacts with and phosphorylates various transcription factors that are critical regulators of cell fate decisions and apoptosis during development. Here we show that lysine 25 of HIPK2 is the major sumoylation site, both in vitro and in vivo, and that the sumoylation of this site occurs in a phosphorylation-dependent manner. This became clear with the finding that kinase-dead HIPK2 (K221R) could not be efficiently sumoylated in vitro. The sumoylation of HIPK2 resulted in the disruption of its interaction with a Groucho corepressor. Consequently, sumoylation inhibited the regulatory activity of HIPK2 on the Groucho-mediated repression of transcription, whereas not on p53-mediated transactivation. These results suggest that phosphorylation-dependent sumoylation enables HIPK2 to drive different target gene transcription by means of differential interactions with its binding partners.