The nucleoporin Nup153 affects spindle checkpoint activity due to an association with Mad1

Abstract

The nucleoporin Nup153 is known to play pivotal roles in nuclear import and exportin interphase cells and as the cell transitions into mitosis, Nup153 is involved in nuclearenvelope breakdown. In this study, we demonstrate that the interaction of Nup153 with thespindle assembly checkpoint protein Mad1 is important in the regulation of the spindlecheckpoint. Overexpression of human Nup153 in HeLa cells leads to the appearance ofmultinucleated cells and induces the formation of multipolar spindles. Importantly, it causesinactivation of the spindle checkpoint due to hypophosphorylation of Mad1. Depletion ofNup153 using RNA interference results in the decline of Mad1 at nuclear pores duringinterphase and more significantly causes a delayed dissociation of Mad1 from kinetochores inmetaphase and an increase in the number of unresolved midbodies. In the absence of Nup153the spindle checkpoint remains active. In vitro studies indicate direct binding of Mad1 to theN-terminal domain of Nup153. Importantly, Nup153 binding to Mad1 affects Mad1'sphosphorylation status, but not its ability to interact with Mad2. Our data suggest thatNup153 levels regulate the localization of Mad1 during the metaphase/anaphase transitionthereby affecting its phoshorylation status and in turn spindle checkpoint activity and mitoticexit.