The CRL4B E3 ubiquitin ligase regulates mitosis by recruiting phospho-specific DCAFs
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Date
2022
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Doctoral Thesis
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Abstract
Ubiquitination is a versatile posttranslational modification best known to target proteins for degradation among other cellular functions. The ubiquitination process is achieved by a cascade of three enzymes with the ubiquitin E3 ligases acting at the final step and ensuring substrate specificity. The Cullin-RING family of E3 ligases (CRLs) ubiquitinate around 20% of the proteome and are shown to have regulatory functions in almost all cellular processes such as development, metabolism, and cell cycle. They are modular platforms with a cullin scaffold, a RING domain containing protein, and specific substrate receptors recruited mainly via an adaptor.
The CRL4 E3 ligases have two paralogs, Cullin-4A (CUL4A) and Cullin-4B (CUL4B), as cullin scaffolds. Both share a high sequence and structural homology of about 80%, except for an extended N-terminal region particularly present in CUL4B. The two CRL4 E3 ligases; CRL4A and CRL4B, are reported to share many redundant functions such as regulation of chromatin remodeling and DNA repair. However, recent studies indicate substrate receptor (DCAFs) preferences for CUL4B or CUL4A despite the shared adaptor DDB1. Interestingly, loss of CUL4B, but not CUL4A, causes embryonic lethality in mice and mutated CUL4B causes X-linked intellectual disability (XLID) in humans.
We observed specific cell cycle defects in CUL4B-depleted cells, but not in CUL4A, during mitosis, implying non-redundant CUL4B functions in cell division. Moreover, we identified that the unique N-terminus of CUL4B is heavily phosphorylated during mitosis, which results in the exclusion of CUL4B from the mitotic chromatin. Interestingly, the XLID disease mutation, CUL4B-P50L, has impaired phosphorylation on a major phospho-site T49. This mutant also exhibits a mitotic delay phenotype, despite equal catalytic ubiquitination activity as WT. Intriguingly, a mass spectrometric approach revealed two potential DCAFs, LIS1 and WDR1, as mitosis-specific and phospho-specific interactors of CUL4B.
LIS1 and WDR1 are mainly unrecognized as substrate receptors (DCAFs), despite of having the characteristic WD40 domain and a helix-loop-helix motifs. Indeed in this work, cellular, and biochemical analysis confirmed the binding of LIS1 and WDR1 to CUL4B via DDB1, which is enhanced by the phosphorylation of the unique N-terminal domain of CUL4B.
Phenotypic characterization of mitotic CUL4B-depleted cells showed enhanced spindle mobility and decreased cortical stiffness, a phenotype also observed for LIS1 depletion. WDR1, on the contrary, only shows a slight reduction in cortical tension, but the mitotic delay phenotype is comparable to CUL4B depletion.
Taken together, we propose that phosphorylated CUL4B assembles novel E3 ligase complexes with the DCAFs LIS1 and WDR1 regulating cytoskeletal dynamics during mitosis consistent with previously reported functions of LIS1 and WDR1.
Further preliminary results strengthened this hypothesis, as several actin regulatory proteins were detected in our mass spectrometry dataset, which could be potential targets for the CRL4BWDR1 E3 ligase.
In addition, we discovered DCTN2, a known interactor of LIS1, as a potential target for the phospho-CRL4B E3 ligase. Moreover, DCTN2 seems to colocalize with CRL4BLIS1 at the centrosome during mitosis.
Intriguingly, not only mutations in CUL4B as well as in LIS1 are implicated in intellectual disability and brain malformation phenotypes. Together with the impaired mitotic phenotype of the disease mutation CUL4B-P50L, we propose an essential function of the phospho-CRL4B E3 ligase in brain development. To test this hypothesis, we analyzed ΔCUL4B forebrain organoids, which show a severe loss of ventricles during forebrain differentiation, similarly to LIS1 patient mutations, indicating a potential link to intellectual disability.
Overall, these results highlight the potential of the extended N-terminus of CUL4B over CUL4A in the context of the newly discovered phospho-regulation of CRL4B during mitosis. Moreover, it raises the question of additional unique functions of CRL4B based on its N-terminus.
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Examiner : Peter, Matthias
Examiner: Kutay, Ulrike
Examiner : Thomä, Nicolas
Examiner : Meraldi, Patrick
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ETH Zurich
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03595 - Peter, Matthias / Peter, Matthias
03543 - Kutay, Ulrike / Kutay, Ulrike