The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we present evidence that human Nek6 is associated with Fe65. Based on the presence of Fe65 WW domain binding motifs ((267)PPLp(270)) in the Nek6 catalytic domain, we observed that Nek6 interacts physically with Fe65 both in vivo and in vitro, using a pull-down approach. Additionally, we detected co-localization of Nek6 and Fc65 via confocal microscopy. Co-localization of Nek6 and Fe65 was disrupted by mutation of the WW domain binding motifs ((267)PPLp(270)). Finally, when transient transfection assays were performed, interaction of Nek6 (wt) with Fe65 induced substantial cell apoptosis, whereas interaction using the Nek6 pplp mutant ((267)PPLp(270) changes (267)APVA(270)) did not. Thus, our observations indicated that Nek6 binds to Fe65 through its (267)PPLp(270) Motif and that the protein-protein interaction between Nek6 and Fe65 regulates their subcellular localization and cell apoptosis. (c) 2007 Elsevier Inc. All rights reserved.