Both DNA replication and centrosome duplication are semi-conservative replication processes that must be coordinated to occur once-and-only-once per cell cycle. Recent findings have suggested that both cyclin dependent kinases and the SCF and APC ubiquitin ligases conspire to regulate both DNA replication and centrosome duplication. Biochemical studies show that the conserved Cdc14 family of phosphatases are selective for dephosphorylating substrates of cyclin-dependent kinases. We have recently discovered that a form of the human Cdc14 phosphatase also has a critical role at the centrosome. Our preliminary studies show that the two known forms of human Cdcl4, called Cdcl4A and Cdcl4B, localize to the centrosome and nucleolus, respectively, We have mapped targeting sequences directing each form to these sites and find that there are sequences that target Cdcl4A and B to their respective sites, but that both proteins partition between centrosomes and nucleoli and that this partition is affected by nuclear import and export signals. Importantly, perturbing the normal function of Cdcl4A by overexpression strongly disrupts centrosome duplication and causes mitotic catastrophe (including chromosome missegregation, a failure to reform nuclei following mitosis, and a block to cytokinesis). Thus, Cdcl4A may play a critical role in mitotic progression and genome stability. Supporting this idea, we find that in Xenopus eggs, the Xenopus Cdcl4 protein is important for mitotic exit. These studies will further test the role of Cdc 14 in mitosis and genome stability. Our specific alms are: (1) to test the role of Cdc 14 and cycin-dependent kinases in specific biochemical steps in the centrosome duplication cycle; (2) to map the centrosomal targeting sequences of Cdcl4A and to use these mutants to determine whether Cdcl4 has additional roles in centrosome maturation or maintenance and later steps in mitosis; (3) to study the specific biochemical requirements for Cdc 14 in mitotic exit using an in vitro system for mitotic exit in Xenopus egg extracts; and (4) to purify and identify Cdcl4A and B interacting proteins using an affinity purification procedure and mass spectrometry to sequence the associated proteins. By purifying proteins associated with both wild type and catalytically inactive (""""""""substrate-trapping"""""""") mutants of Cdcl4, we hope to identify both Cdcl4 regulators and substrates.
These aims will both map Cdc 14-regulated pathways and begin to define the critical Cdk and Cdcl4 targets for regulating steps in mitosis and genome stability.
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