WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR.

The endoplasmic reticulum (ER) is susceptible to wear-and-tear and proteotoxic stress, necessitating its turnover. Here, we show that the N-degron pathway mediates ER-phagy. This autophagic degradation initiates when the transmembrane E3 ligase TRIM13 (also known as RFP2) is ubiquitinated via the lysine 63 (K63) linkage. K63-ubiquitinated TRIM13 recruits p62 (also known as sequestosome-1), whose complex undergoes oligomerization. The oligomerization is induced when the ZZ domain of p62 is bound by the N-terminal arginine (Nt-Arg) of arginylated substrates. Upon activation by the Nt-Arg, oligomerized TRIM13-p62 complexes are separated along with the ER compartments and targeted to autophagosomes, leading to lysosomal degradation. When protein aggregates accumulate within the ER lumen, degradation-resistant autophagic cargoes are co-segregated by ER membranes for lysosomal degradation. We developed synthetic ligands to the p62 ZZ domain that enhance ER-phagy for ER protein quality control and alleviate ER stresses. Our results elucidate the biochemical mechanisms and pharmaceutical means that regulate ER homeostasis.

Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.

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.

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for β propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

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.

Interleukin 33 (IL-33) is an IL-1 family cytokine that plays a central role in immune system by regulating and initiating inflammatory responses. The binding of IL-33 to the suppressor of tumorigenicity 2 (ST2) receptor induces mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) pathways, thereby leading to inflammatory cytokines production in type 2 helper T cells and type 2 innate lymphoid cells. To develop an antibody specific to IL-33 with a defined epitope, we characterized a single-chain antibody variable fragments (scFvs) clone specific to IL-33, C2_2E12, which was selected from a human synthetic library of scFvs using phage display. Affinity (Kd) of C2_2E12 was determined to be 38 nM using enzyme-linked immunosorbent assay. C2_2E12 did not show cross-reactivity toward other interleukin cytokines, including closely related IL-1 family cytokines and unrelated proteins. Mutational scanning analysis revealed that the epitope of IL-33 consisted of residues 149-158 with key residues being L150 and K151 of IL-33. Structural modeling suggested that L150 and K151 residues are important for the interaction of IL-33 with C2_2E12, implicating that C2_2E12 could block the binding of ST2 to IL-33. Pull-down and in-cell assays supported that C2_2E12 can inhibit the IL-33/ST2 signaling axis. These results suggest that the scFv clone characterized here can function as a neutralizing antibody.

Stress granules are membraneless organelles composed of numerous components including ribonucleoproteins. The stress granules are characterized by a dynamic complex assembly in response to various environmental stressors, which has been implicated in the coordinated regulation of diverse biological pathways, to exert a protective role against stress-induced cell death. Here, we show that stress granule formation is induced by morusin, a novel phytochemical displaying antitumor capacity through barely known mechanisms. Morusin-mediated induction of stress granules requires activation of protein kinase R (PKR) and subsequent eIF2α phosphorylation. Notably, genetic inactivation of stress granule formation mediated by G3BP1 knockout sensitized cancer cells to morusin treatment. This protective function against morusin-mediated cell death can be attributed at least in part to the sequestration of receptors for activated C kinase-1 (RACK1) within the stress granules, which reduces caspase-3 activation. Collectively, our study provides biochemical evidence for the role of stress granules in suppressing the antitumor capacity of morusin, proposing that morusin treatment, together with pharmacological inhibition of stress granules, could be an efficient strategy for targeting cancer.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in development and tumor suppression. One of the prominent features of this kinase is that it is tightly regulated by proteasomal degradation. In the present study, we present evidence suggesting that the protein stability of HIPK2 can be regulated by p300-mediated acetylation. p300 increased the protein level of HIPK2 via its acetyltransferase activity. p300 increased the acetylation of HIPK2 while decreased polyubiquitination and its proteasomal degradation. We also observed that DNA damage induced acetylation of HIPK2 along with an increase in the protein amount, which was inhibited by p300 RNAi. Importantly, p300 promoted p53 activation and the HIPK2-mediated suppression of cell proliferation, suggesting acetylation-induced HIPK2 stabilization contributed to the enhanced activation of HIPK2. Overexpression of p300 promoted the HIPK2-mediated suppression of tumor growth in mouse xenograft model as well. Taken together, our data suggest that p300-mediated acetylation of HIPK2 increases the protein stability of HIPK2 and enhances its tumor suppressor function.

Osteoporosis is a disease in which bone mineral density decreases due to abnormal activity of osteoclasts, and is commonly found in post-menopausal women who have decreased levels of female hormones. Sphingosylphosphorylcholine (SPC) is an important biological lipid that can be converted to sphingosine-1-phosphate (S1P) by autotaxin. S1P is known to be involved in osteoclast activation by stimulating osteoblasts, but bone regulation by SPC is not well understood. In this study, we found that SPC strongly inhibits RANKL-induced osteoclast differentiation. SPC-induced inhibitory effects on osteoclast differentiation were not affected by several antagonists of S1P receptors or pertussis toxin, suggesting cell surface receptor independency. However, SPC inhibited RANKL-induced calcineurin activation and subsequent NFATc1 activity, leading to decrease of the expression of Trap and Ctsk. Moreover, we found that bone loss in an experimental osteoporosis mouse model was recovered by SPC injection. SPC also blocked ovariectomy-induced body weight increase and Nfatc1 gene expression in mice. We also found that SPC inhibits RANKL-induced osteoclast differentiation in human macrophages. Since currently available treatments for osteoporosis, such as administration of female hormones or hormone receptor modulators, show serious side effects, SPC has potential as a new agent for osteoporosis treatment.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions in DNA damage response and development. In the present study, we propose that the protein stability and proapoptotic function of HIPK2 are regulated by poly(ADP-ribose) polymerase 1 (PARP1). We present evidence indicating that PARP1 promotes the proteasomal degradation of HIPK2. The tryptophan-glycine-arginine (WGR) domain of PARP1 was necessary and sufficient for the promotion of HIPK2 degradation independently of the PARP1 enzymatic activity. The WGR domain mediated the interaction between HIPK2 and C-terminus of HSP70-interacting protein (CHIP) via HSP70. We found that CHIP can function as a ubiquitin ligase for HIPK2. The interaction between PAPR1 and HIPK2 was weakened following DNA damage. Importantly, PARP1 reduced the HIPK2-mediated p53 phosphorylation, proapoptotic transcriptional activity and cell death. These results suggest that PARP1 can modulate the tumor-suppressing function of HIPK2 by regulating the protein stability of HIPK2.

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.