Biochemical and genetic characterization of cullin 7- and PARC-containing complexes

The ubiquitin-proteasome system controls diverse biological processes, and the cullin family of E3 ubiquitin ligases plays a key role in the ubiquitination of many proteins. Cullin 7 (CUL7), a recently identified cullin, forms a unique SCF-like complex and is required for mouse embryonic development.; To investigate CUL7 function, we sought to identify CUL7-binding proteins, and the p53-associated, Parkin-like Cytoplasmic (PARC) protein, a CUL7 homolog, was identified as a CUL7-interacting protein by mass spectrometry. The hetero- and homodimerization of PARC and CUL7 was confirmed in vivo. To determine the biological role of PARC by itself and in conjunction with CUL7, a targeted deletion of Parc was created in the mouse. In contrast to the neonatal lethality of the Cul7 knockout mice, Parc knockout mice were born at the expected ratios and exhibited no apparent phenotype. Additionally, Parc deletion did not appear to affect the stability, function, or localization of p53.; PARC binds RBX1 and is covalently modified by NEDD8, establishing PARC as a bona fide cullin. Unexpectedly, CUL7 was not found to be neddylated. Examination of complexes containing PARC and CUL7 demonstrated that dimerization of PARC and CUL7 is not dependent on p53 or neddylation status, and the heterodimeric PARC/CUL7 complex does not contain FBXW8. Additionally, p53 can bind heterodimers of PARC/CUL7 and PARC- and CUL7-containing complexes containing markers of activated cullins, such as RBX1 and NEDD8. MudPIT analysis of tandem affinity purified PARC and CUL7 complexes also identified APEX2 as a novel PARC- and CUL7-associated protein.; The potential functional overlap of PARC and CUL7 was addressed by the generation of Parc -/-, Fbxw8 -/- mice. These mice do not show an enhancement of the Fbxw8 -/- phenotype, suggesting non-overlapping functions for PARC and CUL7 in the mouse. Possible genetic interactions of Parc with the p53 pathway were also examined via the Cdkn2a-null mouse model.; Together, these data demonstrate that PARC and CUL7 form homo- and heterodimers and atypical SCF-like complexes, containing novel accessory proteins. Additionally, PARC and CUL7 have no overlapping functions aside from those mediated by the heterodimer, and deletion of Parc has no apparent effect on p53 function.