FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

The E3 Ubiquitin Ligase SCF(Cyclin F) Transmits AKT Signaling to the Cell-Cycle Machinery.

The oncogenic AKT kinase is a key regulator of apoptosis, cell growth, and cell-cycle progression. Despite its important role in proliferation, it remains largely unknown how AKT is mechanistically linked to the cell cycle. We show here that cyclin F, a substrate receptor F-box protein for the SCF (Skp1/Cul1/F-box) family of E3 ubiquitin ligases, is a bona fide AKT substrate. Cyclin F expression oscillates throughout the cell cycle, a rare feature among the 69 human F-box proteins, and all of its known substrates are involved in proliferation. AKT phosphorylation of cyclin F enhances its stability and promotes assembly into productive E3 ligase complexes. Importantly, expression of mutant versions of cyclin F that cannot be phosphorylated by AKT impair cell-cycle entry. Our data suggest that cyclin F transmits mitogen signaling through AKT to the core cell-cycle machinery. This discovery has potential implications for proliferative control in malignancies where AKT is activated.

VprBP/DCAF1 Regulates the Degradation and Nonproteolytic Activation of the Cell Cycle Transcription Factor FoxM1.

The oncogenic transcription factor FoxM1 plays a vital role in cell cycle progression, is activated in numerous human malignancies, and is linked to chromosome instability. We characterize here a cullin 4-based E3 ubiquitin ligase and its substrate receptor, VprBP/DCAF1 (CRL4VprBP), which we show regulate FoxM1 ubiquitylation and degradation. Paradoxically, we also found that the substrate receptor VprBP is a potent FoxM1 activator. VprBP depletion reduces expression of FoxM1 target genes and impairs mitotic entry, whereas ectopic VprBP expression strongly activates a FoxM1 transcriptional reporter. VprBP binding to CRL4 is reduced during mitosis, and our data suggest that VprBP activation of FoxM1 is ligase independent. This implies a nonproteolytic activation mechanism that is reminiscent of, yet distinct from, the ubiquitin-dependent transactivation of the oncoprotein Myc by other E3s. Significantly, VprBP protein levels were upregulated in high-grade serous ovarian patient tumors, where the FoxM1 signature is amplified. These data suggest that FoxM1 abundance and activity are controlled by VprBP and highlight the functional repurposing of E3 ligase substrate receptors independent of the ubiquitin system.

APC/C and SCF(cyclin F) Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry.

The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.

PIDD death-domain phosphorylation by ATM controls prodeath versus prosurvival PIDDosome signaling.

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.