Centrosomes organize the microtubule cytoskeleton and transform into the spindle poles for segregation of chromosomes during mitosis. Abnormal centrosomes produce aneuploidy in human cancers and cause other human sensory and developmental disorders. This project has a long- standing focus on protein phosphatase-1 (PP1) and inhibitor-2 (Inh2) that localize to centrosomes and regulate multiple centrosomal enzymes. Knockdown of Inh2 by RNAi in human cells causes lagging mitotic chromosomes, failure of cytokinesis and formation of multinucleated cells with supernumerary centrosomes, like seen in human tumors. The phenotype is rescued by co-expression of Inh2 that avoids the RNAi knockdown. In Drosophila there is a single Inh2 gene that is maternally expressed in oocytes and embryos during early stages of development. Flies hypomorphic for Inh2 have increased embryonic lethality, with severely reduced hatch rates. Surviving embryos exhibit loss of syncytial mitotic synchrony and have many bridged nuclei due to faulty chromosome segregation. Embryo survival is rescued by dose-dependent transgenic expression of D-Inh2. Thus, results with both human and Drosophila support a key role for Inh2 in mitotic chromosome segregation.
Specific Aim 1 seeks to define mechanisms by examining the effects of Inh2 on Aurora B activation and phosphorylation of mitotic substrates that mediate proper chromosome alignment at metaphase. Inh2 is phosphorylated during mitosis at a conserved PxTP site by CDK1::cyclinB1. This phosphorylation disrupts Inh2 binding to the prolyl isomerase Pin1, which drastically alters Pin1 substrate specificity with mitotic phosphoproteins.
Specific Aim 2 will determine the structure of Inh2::Pin1 by NMR and define effects of phosphorylation on the interactions and the binding of substrates.
Specific Aim 3 will use rescue of RNAi knockdown cells and hypomorphic Drosophila to dissect the multifunctional nature of Inh2 as a PP1 inhibitor, mitotic kinase activator and prolyl isomerase regulator. Mitotic kinases are already designated targets for drug development by the pharmaceutical industry, but regulation by PP1 and Inh2 has heretofore been underappreciated. Effective diagnosis and treatment of human diseases and accurate monitoring of clinical efficacy of new targeted therapies will need to incorporate information from this unique project.
Human sensory responses such as sight, hearing and smell depend on cilia on the surface of epithelial tissues. These cilia are rooted at cellular structures called centrosomes. Centrosomes fulfill multiple functions besides supporting the cilia, such as control of cell division and separation of chromosomes during division. Defects in centrosomes can result in a variety of human diseases including loss of sight and smell, and especially cancer, where too many centrosomes are seen in tumor cells that scatter chromosomes. This project studies a protein found in all animals that is critical and necessary for proper centrosome function. The goal is to understand how this protein acts to connect multiple signaling inputs into centrosome actions. Understanding cellular mechanisms is the basis for diagnosis and treatment of human diseases.
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