This is a Competitive Revision for GM50403 of Notice Number (NOT-OD-09-058), and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The state of Michigan has been particularly hard hit by the economic crisis. Moreover, last year Pfizer closed its largest research campus;located here in Ann Arbor. This resulted in the termination of over 2000 highly skilled researchers, many of whom remain unemployed. This application seeks to employ three of these investigators, and also to expand our studies of the parent grant. The overall goal of the parent grant is to study phosphatidylinositol (3,5)-bisphosphate (PI3,5P2), a very low abundance, and relatively unstudied signaling lipid. PI3,5P2 is found in all eukaryotes, from yeast to humans. We found that depletion of PI3,5P2 leads to perinatal lethality and massive neurodegeneration in mice. Moreover, minor defects in the PI3,5P2 signaling pathway are found in 2% of ALS patients. The focus of the parent grant is to study PI3,5P2 in yeast, in the hopes that these studies will reveal insights into PI3,5P2 function in humans as well.
The Aims of the parent grant are 1) determine how levels of PI3,5P2 are regulated in yeast, and 2) determine proteins that are downstream effectors of PI3,5P2 in yeast. As part of Aim 2, we uncovered over 270 PI3,5P2 candidate binding proteins. We are currently devising the best approaches to determine which candidates are bona fide downstream effectors of PI3,5P2. The current candidates represent a large number of endosomal related events including proteins involved in multiple membrane trafficking steps, and also subunits of the vacuolar ATPase, other transporters and a significant number of uncharacterized proteins. Our goal is to define which pathways require PI3,5P2 for their function(s). Here we seek to expand these studies and follow up on a set of new and unexpected candidates. We found that a major cell regulator, the TOR kinase, as well as the TORC1 regulator, Kog1/Raptor, bind specifically and avidly to PI3,5P2. Importantly, Dr. Alan Saltiel and co-workers (U. Michigan) have independent evidence that mammalian Raptor binds PI3,5P2;and that PI3,5P2 is required for the activation of Tor1 in adipocytes. Thus, we propose to expand our current studies and to perform the following three aims. 1) Identify novel downstream targets of TORC1, 2) Determine whether PI3,5P2 is upstream and/or downstream of Tor1 function, and 3) Determine each site on Tor1 and Kog1 that bind PI3,5P2, and also test whether Gtr1/Gtr2 (RagA-D) directly bind PI3,5P2.
The overall goals of this application, are to 1) create employment opportunities for skilled scientists, and 2) expand the goals of the parent grant. We recently made the unexpected finding that TORC1, a key cellular regulator of critical medical importance, binds specifically and avidly to the signaling lipid PI3,5P2. We will exploit these findings to pursue new avenues to determine how TORC1 is regulated, and to uncover novel downstream TORC1 targets.
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|Jin, Natsuko; Jin, Yui; Weisman, Lois S (2017) Early protection to stress mediated by CDK-dependent PI3,5P2 signaling from the vacuole/lysosome. J Cell Biol 216:2075-2090|
|Kim, Seong M; Roy, Saurabh G; Chen, Bin et al. (2016) Targeting cancer metabolism by simultaneously disrupting parallel nutrient access pathways. J Clin Invest 126:4088-4102|
|Jin, Natsuko; Lang, Michael J; Weisman, Lois S (2016) Phosphatidylinositol 3,5-bisphosphate: regulation of cellular events in space and time. Biochem Soc Trans 44:177-84|
|Jin, Yui; Strunk, Bethany S; Weisman, Lois S (2015) Close encounters of the lysosome-peroxisome kind. Cell 161:197-8|
|McCartney, Amber J; Zolov, Sergey N; Kauffman, Emily J et al. (2014) Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression. Proc Natl Acad Sci U S A 111:E4896-905|
|Li, Sheena Claire; Diakov, Theodore T; Xu, Tao et al. (2014) The signaling lipid PI(3,5)P? stabilizes V?-V(o) sector interactions and activates the V-ATPase. Mol Biol Cell 25:1251-62|
|McCartney, Amber J; Zhang, Yanling; Weisman, Lois S (2014) Phosphatidylinositol 3,5-bisphosphate: low abundance, high significance. Bioessays 36:52-64|
|Jin, Natsuko; Mao, Kai; Jin, Yui et al. (2014) Roles for PI(3,5)P2 in nutrient sensing through TORC1. Mol Biol Cell 25:1171-85|
|Li, Xinran; Wang, Xiang; Zhang, Xiaoli et al. (2013) Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics. Proc Natl Acad Sci U S A 110:21165-70|
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