RT Journal Article
SR Electronic
T1 Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e202201472
DO 10.26508/lsa.202201472
VO 5
IS 12
A1 Dominika Kowalczyk
A1 Mark A Nakasone
A1 Brian O Smith
A1 Danny T Huang
YR 2022
UL https://www.life-science-alliance.org/content/5/12/e202201472.abstract
AB ARF tumor suppressor protein is a key regulator of the MDM2-p53 signaling axis. ARF interferes with MDM2-mediated ubiquitination and degradation of p53 by sequestering MDM2 in the nucleolus and preventing MDM2-p53 interaction and nuclear export of p53. Moreover, ARF also directly inhibits MDM2 ubiquitin ligase (E3) activity, but the mechanism remains elusive. Here, we apply nuclear magnetic resonance and biochemical analyses to uncover the mechanism of ARF-mediated inhibition of MDM2 E3 activity. We show that MDM2 acidic and zinc finger domains (AD-ZnF) form a weak intramolecular interaction with the RING domain, where the binding site overlaps with the E2∼ubiquitin binding surface and thereby partially reduces MDM2 E3 activity. Binding of human N-terminal 32 residues of p14ARF to the acidic domain of MDM2 strengthens the AD-ZnF-RING domain interaction. Furthermore, the N-terminal RxFxV motifs of p14ARF participate directly in the MDM2 RING domain interaction. This bivalent binding mode of p14ARF to MDM2 acidic and RING domains restricts E2∼ubiquitin recruitment and massively hinders MDM2 E3 activity. These findings elucidate the mechanism by which ARF inhibits MDM2 E3 activity.