Novel functions of the von Hippel-Lindau tumor suppressor

In the research presented in this thesis we studied possible tumor suppressing activities of pVHL. A general introduction about tumor suppressing activities of the von Hippel-Lindau (VHL) tumor suppressor is summarized in Chapter 1. In Chapter 2, we identified that expression and activity of the transcription factor E2F1 is negatively regulated by pVHL in a HIFalpha-independent manner. The majority (around 90%) of clear-cell RCC samples from VHL patients show high nuclear E2F1 staining, suggesting that E2F1 might be involved in tumorigenesis in patients with VHL disease. The alpha-domain of pVHL seems to be essential for E2F1 regulation in RCC cells, however does not affect cell cycle progression of asynchronous RCC cells. In Chapter 3, we give an overview of recent literature linking ciliary components and signaling pathways with tumorigenesis. In Chapter 4, we focus on the role of pVHL in the regulation of primary cilia. We show that pVHL is able to maintain primary cilia and ciliary function of kidney epithelial cells, in a HIFalpha-independent fashion. In these cells we show axonemal localization of pVHL. However, VHL -type 2A- disease associated allele Y112H and pVHL lacking the microtubule-binding domain, both known for their inability to protect stabilized microtubules from nocodazole treatment, are no longer able to localize along the ciliary axoneme or to maintain functional primary cilia in cultured kidney epithelial cells. Surprisingly, pVHL19 was not observed to localize to the primary cilium nor could it restore cilia function as measured by calcium influx. In Chapter 5, we searched for the missing mechanism for pVHL’s ciliary function. Applying yeast-two-hybrid assays, we identified an interaction between pVHL and KIF3A, a subunit of the anterograde kinesin-2 motorcomplex, shown to be important in correct intraflagellar transport (IFT) and ciliogenesis. We show that pVHL interacts endogenously with all members of kinesin-2 and we identify two domains to be important for binding kinesin-2. The N-terminal domain of pVHL binds KAP3 and the microtubule-binding domain binds KIF3A. Interestingly, VHL variant alleles displaying the inability to stabilize microtubules or to maintain primary cilia have a disrupted binding to kinesin-2, suggesting that kinesin-2 interaction is essential for pVHL to stabilize microtubules and/or to maintain primary cilia. In Chapter 6 we show that cytoplasmic mobility and subcellular localization of pVHL is dependent on the presence of ATP. Removing ATP does result in centrosomal localization of immobile pVHL. However, VHL variant alleles displaying a disrupted binding to kinesin-2 are less responsive to ATP-depletion. Furthermore, inhibiting pVHL-KIF3A interaction by means of a dominant negative form of KAP3 (deltaN-KAP3) or KIF3A RNAi increases pVHL mobility, indicating that KIF3A affects the mobility of pVHL. Finally, in Chapter 7 we give a summarizing discussion of the work described in this thesis and its implications