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Abstract

Centrosomes are important cell organelles that serve as main microtubule-organizing centers and are involved in diverse cellular processes, most importantly cell division by facilitating formation of the bipolar spindle in mitosis. Each centrosome consists of a pair of centrioles, which is duplicated exactly once per cell cycle. The serine/threonine protein kinase polo-like kinase 4, PLK4, is known as the master regulator of centriole duplication. Overexpression of PLK4 is sufficient to induce tumor formation in mice, by causing centrosome amplification and chromosome missegregation in mitosis as a source for genomic instability. In order to prevent centriole overduplication, PLK4 protein levels are tightly regulated by ubiquitylation and proteasomal degradation. Protein ubiquitylation is a post-translational modification, carried out by an enzymatic cascade consisting of three enzymes: E1, E2 and E3. The SKP1-CUL1-b-TrCP E3 ubiquitin ligase complex has been shown to recognize the substrate PLK4 upon trans-autophosphorylation of a degron motif and regulate its protein levels by ubiquitylation and proteasomal degradation. However, a b-TrCP binding mutant of PLK4 has been found to be still ubiquitylated and partly degraded, indicating that the exact regulation of PLK4 protein levels has not been unraveled entirely yet. In the presented thesis, I identified PLK4 as a novel substrate of the E3 ubiquitin ligase CUL4-DDB1-DCAF1, CRL4DCAF1, which ubiquitylates and thereby targets PLK4 for degradation in G2 phase of the cell cycle to prevent premature centriole duplication in mitosis. DCAF1 serves as a substrate binding domain of the complex, which I showed to bind PLK4 in a phosphorylation-independent manner. Overexpression of DCAF1 enhanced the ubiquitylation of PLK4, while knockdown of DCAF1 increased PLK4 protein levels and caused the formation of multipolar spindles in mitosis. I found that the regulation of PLK4 by CRL4DCAF1 also affects the interaction between PLK4 and its substrate STIL, as well as the process of centriole disengagement at the onset of centriole biogenesis. Taken together, I identified a new mechanism for regulating PLK4 protein levels in centriole duplication that is dependent on the CRL4DCAF1 ubiquitin ligase complex. My results contribute to a better understanding of the complex regulation and might open up new possibilities to target deregulated or overexpressed PLK4 as a novel approach for cancer therapy.