Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation

Abstract

SUMMARY Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation.

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development

“…Consistent with this observation, both ubc-18 and ubc-3 regulate in vivo steady-state levels of SUP-36 protein; however, the "Ub priming" function of ARI-1 is not required for other Cullin E3s in C. elegans. Our findings complement a recent study in mammalian cell culture suggesting that RBR and CRL E3 complexes can work together (9). Furthermore, our work provides biological evidence for the existence of crosstalk between two different types of E3s, RING-and RBR-type E3s, and their respective, dedicated E2s in an organism.…”

“…Although our results are in some ways reminiscent of those from a recent study in mammalian cell culture suggesting that RBR and CRL E3 complexes can work together (9), there are some notable distinctions. Our findings indicate that the "Ub priming" function of ARI-1 is not required for all CUL-1 substrates or for other Cullin E3s in C. elegans.…”

“…4D, lanes 8/9 compared with lanes 2/3). This observation can be explained by N8-CUL-1 binding and activating autoinhibited HHARI, as reported recently (9,31,32). Second, N8-CUL-1 bound directly to SUP-36 ( Fig.…”

“…A study published while the present paper was in preparation demonstrated that depletion of HHARI in cell culture leads to accumulation of known CRL substrates, and that some CRL substrates are primed with Ub by HHARI/UBCH7 followed by poly-Ub chain formation by CDC34/CRL in vitro (9). Although our genetic and biochemical evidence complements those findings by showing that the orthologs of these enzymes regulate the levels of a common substrate in worms and work together to ubiquitinate that common substrate in vitro, our genetic data also imply that the ability of the E2/E3 pairs to work coordinately is not critical for all biological CUL-1-dependent processes.…”

“…Notably, our biochemical reactions were carried out in the absence of either (human) SKP1 or an F-box protein. To rule out nonspecific modification of SUP-36 by UBCH7/CUL-1/N8-CUL-1, we performed a biochemical assay analogous to those previously published for canonical Cullin substrate ubiquitination in the presence of UBCH7/HHARI, in which precharged UBCH7∼Ub is added to initiate reactions (9). In these reactions (Fig.…”