The proliferation of cells depends on the duplication and segregation of their genomes. The latter is an immensely complex process that remains poorly understood at a molecular level. Mistakes during mitosis contribute to cancer whereas mistakes during meiosis that cause aneuploidy are the leading cause of infertility and mental retardation. During mitosis, sister DNA molecules are dragged towards opposite poles of the cell due to their prior attachment to microtubules with opposite orientations (bi-orientation). Bi-orientation involves dissolution of the nuclear membrane, changes in chromosome organization, and re-organization of the spindle apparatus. How mitotic cells coordinate these disparate but inter-locking processes is poorly understood. One thing, however, is certain. Protein kinases like Cdk1 have fundamental roles. Nevertheless, Cdk1's actual function remains mysterious despite recognition of its importance by a Nobel prize. The same is true for other mitotic kinases, such as Plk1 and Aurora A and B. We need to know what set of proteins are phosphorylated, what their functions are, or how phosphorylation changes their activity. Identification of kinase substrates has been hampered by difficulties in mapping phosphorylation sites, in experimentally controlling protein kinase activity, and in evaluating the physiological consequences of defined phosphorylation sites. The premise behind this proposal is that all three hurdles can be overcome by new technologies, namely the use of RNA interference to identify in a systematic (functional genomics) manner potential substrates, iTAP- tagging to purify protein complexes, small molecules to inhibit specific kinases in a controlled fashion, and mass spectrometry to identify phosphorylation sites on complex subunits. Because the concept behind this project could be applied to other areas, it will have an impact on European cell biology far beyond the cell cycle community.