Phosphoinositide Signaling in Cytosol and Nucleus

University Of Wisconsin-Madison, Madison WI

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Abstract

Project Summary The research proposed seeks to reveal mechanisms for spatial phosphoinositide (PI) signaling in the cytosol and nucleus. These pathways have broad implications for cancer, neurodegenerative disorders, and other diseases. PI signaling generates seven distinct polyphosphoinositide (PIPn) messengers. EGF and other agonists activate the class I PI 3-kinase (PI3K)/Akt pathway on membranes that controls cell growth and other key cellular functions. In nuclei, PIPn pathways are separate from membranes, controlling stress responses that impact DNA repair, cell survival, and other critical events. The work on agonist-activated PI3K signaling is focused on the spatial and temporal activation of PI3K, which generates PIP3 to activate Akt and other pathways. The PI3K pathway plays a key role in cancer both by agonist activation and by PI3K mutations that hyperactivate the pathway independent of agonists. We show that the PI3K pathway is incorporated into an IQGAP1 complex, which uses PI to sequentially synthesize PI4P, PIP2 and PIP3, the latter activating mTORC2, PDK1 and Akt. The utilization of PI suggests that the IQGAP1 complex may function at any cytoplasmic membrane, but dogma states that PI3K signaling occurs at the plasma membrane. We have shown that agonist-stimulated PI3K signaling is endosomal and is regulated by interactions with MAP4 and PI3P that spatially targets PI3K to microtubules and endosomes, respectively. PI3K interactions with MAP4 and PI3P are required for agonist-stimulated signaling. However, the underlying mechanisms for the assembly of PI3K in these complexes is unknown. Although constitutively active PI3K mutants that hyperactivate the pathway in cancer still requires IQGAP1, MAP4 and PI3P interactions, the mechanisms by which these PI3K mutants stimulate the assembly of these complexes needs to be defined. In contrast to the cytosolic pathway, we have shown that Nuclear PI signaling is separate from membranes and is mediated by linking PIPns (likely covalently) to PIPn effector proteins (PIPylation) including p53. PIPns are linked to p53 and sequentially phosphorylated to generate a p53-PIP3 complex that activates nuclear Akt by assembly of mTORC2, PDK1 and Akt. Remarkably, wild-type (p53wt) and mutant p53 (p53mt) regulate Akt activation differently, suggesting a mechanism for its tumor suppressor and oncogenic functions, respectively. Notably, PIPns are linked to many nuclear proteins, indicating a broader “PIPylome”. Remaining questions are the chemical nature and enzymatic mechanism of the PIPn linkage, the identification of the cellular PIPylome, and the regulation of PIPylated proteins by PIPns. We have also discovered that PI transfer protein (PITP)s and PI 4-kinase II (PI4KII) are necessary for PIPylating proteins but the mechanisms are unknown. Moreover, PIPns are linked to multiple DNA repair proteins and the functional role of PIP linkage in the DNA damage response will be investigated. Significance- The scaffolded endosomal PI3K activation and PIPylation of cellular proteins are paradigm-shifting and have vast implications for biology and therapeutics.