| Yes-associated protein (YAP) is a co-transcription factor that acts downstream of the evolutionarily conserved Hippo pathway. Canonically, this pathway regulates tissue growth in flies and mammals, by controlling the nuclear localization of YAP. Interestingly, in addition to the conserved functions of this pathway, some of the mammalian orthologs of pathway components (e.g. MST, RASSF1, WW45, and LATS) have been shown to localize to the nucleus and alterations in their expression induces alterations in mitotic processes, suggesting additional roles for these proteins in mitosis. In this thesis, I have uncovered a role for the Hippo pathway effector protein, YAP, in cytokinesis. YAP was found to localize to the central spindle and cytokinetic midbody and biochemical analysis demonstrated that YAP is phosphorylated by the mitotic regulatory kinase CDK1 during mitosis. Time-lapse microscopy of cells in which YAP was downregulated by shRNA revealed that reduction in YAP expression causes a delay in abscission and induces a cytokinesis phenotype associated with increased contractile force, membrane blebbing and bulges, and abnormal spindle orientation; consequently, this leads to an increased frequency of multinucleation, micronuclei, and aneuploidy. Expression of or expression of a variant of YAP that could not be phosphorylated at the mitotic phosphoacceptor sites induced a phenotype similar to that of YAP knockdown, suggesting that mitotic YAP phosphorylation is critical for YAP's function in cytokinesis. Reduction in YAP expression also disrupted the localization of ECT2, MgcRacGap, Anillin, and RHOA, proteins important for cleavage furrow function during cytokinesis, Reduction of YAP also increased levels of phosphorylated myosin light chain, which activates myosin II contractile activity. These findings suggest that YAP is required for proper coordination of these contractile processes involved in cytokinesis. In addition, the YAP mitotic phosphorylation sites are required for interaction with the scaffold polarity protein PATJ, and PATJ co-localizes with YAP at the cytokinesis midbody. PATJ knockdown induces cytokinesis defects and spindle orientation alterations similar to those detected in YAP- depleted cells or cells expressing a non-phosphorylatable mutant of YAP. This study reveals an unanticipated role for YAP during mitosis and implicates YAP in processes that control the proper organization of cytokinesis machinery through interaction with the polarity protein PATJ. Thus, these studies demonstrate a previously unanticipated role for YAP that is independent of its activity as a transcriptional coactivator. In addition, although YAP is known to function as a potent oncogene, our findings indicate that YAP may also act as a tumor suppressor in certain contexts since loss of YAP could lead to genetic alterations associated with defective cytokinesis. These studies add to the complexity of YAP regulation in cancer as well as in normal development and provide a framework for future studies in a new area of Hippo pathway biology. |
