Novel Roles of Cullin-RING Ligases in Cell Signalling and Implications in Health and Disease

Cullin-RING ligases (CRLs) play fundamental functions in key physiological and pathological processes. To identify novel roles of CRLs in cell signalling and their implication in health and diseases, we performed two separate studies: In study 1, we analysed genomic databases to identify CRLs that are hypermutated in cancer. We found that the CRL substrate receptors FBXO24 and DCAF12L2 are hypermutated at critical domains that are necessary for the proper structure/function of the CRL1FBXO24 and CRL4DCAF12L2 complexes, respectively. We showed that the FBXO24(T65P) mutation within the F-box domain as found in cancer disrupts the CRL1FBXO24 complex by blocking the binding of FBXO24 to SKP1. Similarly, we found that DCAF12L2 is hypermutated in cancer at the amino acids sequence positions 334, 335, and 337 within the WD40 repeats that mediate substrate binding. Our affinity purification coupled with mass spectrometry analysis identified MEKK4 and the WDR11 complex as two independent substrates of CRL4DCAF12L2. Moreover, the DCAF12L2(P334L), (R335C), and (R335H) mutations block DCAF12L2 binding to MEKK4 and the WDR11 complex. We also showed that FAM91A1, a component of the WDR11 complex, is ubiquitylated by CRL4DCAF12L2 and proposed that the ubiquitylation of FAM91A1 might be critical in regulating the stability and function of the WDR11 complex. In study 2, we employed a proteomic approach to identify CRL3 complexes activated at the cellular membrane. Our mass spectrometry analysis identified CRL3KLHL12 as a ubiquitin ligase that mediates the ubiquitylation of Lunapark, an endoplasmic reticulum (ER) shaping protein. We show that Lunapark interacts with mechanistic target of rapamycin complex1 (mTORC1) and that the ubiquitylation of Lunapark regulates mTORC1 activation. In addition, we show that the inhibition of Lunapark ubiquitylation leads to neurodevelopmental defects.