The Neks are kinases similar to NIMA, which is essential for mitosis in the fungus Aspergilus nidulans.. In mammals, there are 11 Neks, but so far only only Nek2, Nek6, Nek7 and Nek9 have been directly implicated in mitotic regulation. Recent studies support the idea that Nek6, Nek7 and Nek9 act together in the same signaling cascade in mitosis and that Nek6 and Nek7 have an independent role in mitotic spindle formation and cytokinesis. However, little is known about the mechanism whereby Nek6, Nek7 and Nek9 may contribute to different events in mitotic progression or other functions in the cell. Recent studies showed the involvement of the protein Nek7 in mechanisms of centrosome duplication, and besides this, the over-expression of Nek7 in breast, colorectal, laryngeal, and lung cancer as well as in non-Hodgin lymphoma. Thus, the employment of functional studies is necessary to clarify the function of Nek7 in carcinogenesis, and its role in the signaling pathway, , cell cycle and centrosome function. Here we will perform: functional studies, involving silencing RNA (iRNA) of Nek7, its over-expression in human cells and the identification of proteins and possibly substrates that interact with Nek7 through pull down assays, immunoprecipitation, followed by mass spectrometry in an attempt to elucidate the signaling cascade which the Nek7 is involved. Previous yeast-two hybrid studies in our laboratory identified an interaction between Nek7 and MAT1. Here we will be perform ate experiments to confirm this interaction and to identify the functional consequences of this interaction . MAT1 makes part of a regulatory triad with cyclin H and cdk7 (called CAK) that together participate in the the cell cycle: initiation of DNA synthesis in the S phase and the cells entry into mitosis as well as in events related to tumorigenesis. We will study the activity of CAK (CDK7/Ciclina H/MAT1) in MEFs (Mouse Embryonic Fibroblast) derived from embryonic mice Nek7-/ - or in cells with iRNA knock down of Nek7 or its protein super-expression (Flag-Nek7). We will study tumor growth or growth inhibition and CAK activity and phosphorylation in mice injected with cells with NEK7 siRNA k.o., Nek7 kinase dead and Nek7 phosphomimeti. Finally, in vitro bioassays will be performed, using the HTS platform (High Throughput Screening) newly installed in LNBio for screening and characterization of inhibitors Nek7 that can be used for inhibition tests of tumor growth.
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