All cells are regulated by the phosphoinositide cycle (PI-cycle). In the PI cycle, phosphatidylinositol is sequentially phosphorylated, first on the fourth hydroxyl of the myo-inositol ring, and then by the phosphatidylinositol-4-phosphate 5-kinases (PIP 5-kinase), forming phosphatidylinositol-4,5-biphosphate (PIP2). PIP2 occupies an essential position in the PI cycle; it is the precursor for all of the PI-derived second messengers. This suggests that the PIP 5-kinases are highly regulated enzymes. Further, PIP2 is required for cell proliferation in yeast and mammalian cells. The type I and II PIP 5-kinases are sequence- distinct enzymes and regulated by different mechanisms. Antibodies to these enzymes are specific and not crossreactive. In established cell lines, there appear to be single isoforms of the type I and II PIP kinases by Western blotting. Indirect immunofluorescence staining of cells with the antibodies shows that both kinases are located on cytosolic membranes. However, a fraction of the staining for both PIP 5-kinases is intensely located to sites of active DNA replication. These data suggest a specific or direct role for the PIP 5-kinases in regulation of DNA synthesis or S-phage progression. The proposed work will critically assess this hypothesis with the following Specific Aims: (1) To establish the cellular location of the PIP 5-kinases with an emphasis on the subnuclear location to sites of DNA replication. Using established cell lines and mouse oocytes, studies will be initiated to determine if PIP 5-kinases are required for DNA replication or S-phase progression. (2) To determine the stimuli which localize type I and II PIP kinases to nuclei and sites DNA replication. Do growth factors or other mitogens regulate intracellular localization of the PIP 5-kinases? Which PIP kinases are regulated by EGF receptor activation. (3) To determine which PIP 5-kinase isoforms are associated with focal adhesion sites and the role of PIP 5-kinases in integrin receptor signalling. (4) To isolate and sequence the cDNAs for the type I and II PIP 5-kinases. Using cellular and biochemical techniques, the PIP 5-kinases will be localized within nuclei during the cell cycle. The signals for nuclear location of the PIP kinases will be studied. Using the isolated cDNA, we will explore the possibility that the PIP kinases interact with SH-3 domains. A direct role for the PI cycle in nuclear signaling and specifically DNA replication has many implications for proliferative diseases and cancer.
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