Cullin 3 targets the tumor suppressor gene ARMC5 for ubiquitination and degradation

in Endocrine-Related Cancer

Correspondence should be addressed to I P Cavalcante or B Ragazzon: isadoracavalcante@gmail.com or bruno.ragazzon@inserm.fr

*(A Vaczlavik and L Drougat contributed equally to this work)

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ARMC5 (Armadillo repeat containing 5 gene) was identified as a new tumor suppressor gene responsible for hereditary adrenocortical tumors and meningiomas. ARMC5 is ubiquitously expressed and encodes a protein which contains a N-terminal Armadillo repeat domain and a C-terminal BTB (Bric-a-Brac, Tramtrack and Broad-complex) domain, both docking platforms for numerous proteins. At present, expression regulation and mechanisms of action of ARMC5 are almost unknown. In this study, we showed that ARMC5 interacts with CUL3 requiring its BTB domain. This interaction leads to ARMC5 ubiquitination and further degradation by the proteasome. ARMC5 alters cell cycle (G1/S phases and cyclin E accumulation) and this effect is blocked by CUL3. Moreover, missense mutants in the BTB domain of ARMC5, identified in patients with multiple adrenocortical tumors, are neither able to interact and be degraded by CUL3/proteasome nor alter cell cycle. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives in the understanding of its tumor suppressor activity.

Supplementary Materials

    • Figure S1 - ARMC5 regulates cell cycle and cyclin E turnover in HEK293 cells Propidium iodide was used to determine DNA content. (A) Flow cytometry analysis after ARMC5 depletion revealed a decrease in the percentage of cells in G1 phase and an increase in S phase. (B) Depletion of ARMC5 led to an increase in full length (FL) and low molecular weight (LMW) cyclin E. (C) Overexpression of WT ARMC5 increases the number of cells in G1 phase. However, co-expression of WT ARMC5 and CUL3, as well as overexpression of p.L754P mutated ARMC5 have no longer an effect in cell cycle. Images are representative of at least three independent experiments. Significance was assessed by using two-way ANOVA, followed by Bonferroni post-test.
    • Figure S2 - ARMC5 depletion increases CCNE1 mRNA transcription in both (A) H295R and (B) HEK293 cells. Significance was assessed by using student’s t-test.
    • Figure S3 - ARMC5 depletion favours cell cycle progression in H295R cells Propidium iodide was used to determine DNA content. (A) Flow cytometry analysis after ARMC5 depletion. (B) Synchronization of cells in late G1 phase with aphidicolin (10µM) for 24h. (C) Flow cytometry analysis 4h (C), 8h (D), 12h (E) and 24h (F) after release from aphidicolin. Significance was assessed by using two-way ANOVA, followed by Bonferroni post-test.
    • Supplemental table 1