Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

Abstract

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1-3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN-CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4-CSN complex, thereby regulating nucleotide excision repair and cell death. Inositol pyrophosphates containing seven (IP7) or more phosphate groups on a myo-inositol ring are synthesized from inositol hexakisphosphate (IP6) primarily by a family of IP6 kinases that are conserved from yeast to humans and mediate diverse physiologic functions (1, 2). Among the three mammalian IP6K isoforms, IP6K1 and IP6K2 are widely distributed, whereas IP6K3 is expressed primarily in the brain (3). IP6K1 plays a role in diabetes (4), DNA homologous recombination (HR) repair (5), spermatogenesis (6), and chromatin modifications (7).The Cullin-RING ubiquitin ligases (CRLs) control fundamental biological processes by mediating 20% of ubiquitindependent protein turnover (8). These E3 ligases are multiprotein complexes composed of the scaffold Cullins (Cul 1-7), the E2-interacting RING-finger protein Roc1/2, adaptor proteins specific for each Cullin family member, and adaptor-interacting substrate receptors that target substrates for ubiquitylation. CRL4, a DNA-damage-sensing member of this family (9), mediates the degradation of numerous substrates involved in cell cycle regulation (CDT1, p21, p27) as well as cell growth or death (c-Jun, p53) and is aberrantly active in many tumor types (10). The CRL4 complex comprises Cul4A/B and the adaptor protein damagespecific DNA binding protein 1 (DDB1). DDB1 binds to a family of WD40 domain-containing proteins that are substrate receptors (11, 12). The complex of DDB1 and its substrate receptor DDB2 binds directly to UV-damaged DNA to initiate nucleotide excision repair (NER) by ubiquitylating local histones and the NER machinery (9). Loss of DDB activity results in group E xeroderma pigmentosum, whose victims are hypersensitive to UV light. However, it remains unclear how CRL4 is activated by UV.In plants, CRL4 is also regulated by UV. Thus, UV absorption by tryptophan residues in the dimer interface of UV-B Resistance 8 (UVR8) triggers dimer dissociation (13). Monomeric UVR8 binds and seque...

Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

“…To examine whether CSN2's N-terminal tail is critical for physiologic cullin-CSN assembly, we conducted rescue experiments using cells with CSN2 stably depleted as previously reported (21). In these cells, binding of cullins to other CSN subunits, such as CSN5, is markedly reduced (Fig.…”

“…Furthermore, this interaction is disrupted by UV radiation, leading to CRL activation (21). In contrast to IP6K1, IP5K binding to Cul4A is not altered upon UV stimulation (Fig.…”

“…We previously reported that the IP6 metabolizing enzyme IP6K1 binds to CRL4A to promote the formation of the inactive CRL4A-CSN complex (21). Furthermore, this interaction is disrupted by UV radiation, leading to CRL activation (21).…”

“…IP6 can be further phosphorylated by IP6 kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to generate highly energetic inositol pyrophosphate species (IP7 and IP8) (20). Recently, we reported that IP6 kinase-1 (IP6K1) binds both CSN and CRL4A to promote CRL4A-CSN complex formation in a catalytic activity-independent manner (21). Both kinase-dead IP6K1 and the IP6K inhibitor TNP [N(2)-(m-(trifluoromethy)lbenzyl) N(6)-(p-nitrobenzyl)purine] further stabilize the CRL4A-CSN complex, suggesting that the catalytic activity of IP6K1 (i.e., the turnover of IP6), negatively regulates CRL4A-CSN complex stability.…”

Lentinula edodes Genome Survey and Postharvest Transcriptome Analysis

“…InsP6K forms diphosphoinositol pentakisphosphate [InsP (7)], which induces autophagy (67). Furthermore, InsP6K mediates the assembly/disassembly of the cullin-RING ubiquitin ligases (CRL)-signalosome complex to regulate cell death (68). The vacuolar assembling sorting protein VPS16 (g9687.t2) (Table S7), which is involved in vacuolar assembling for biosynthetic, endocytic, and autophagic pathways (69), was also upregulated after harvest.…”