Phosphoinositides (PIs) play critical roles in mediating a wide range of cellular responses to multiple hormones and growth factors. PI-modifying enzymes, such as kinases and phosphatases, along with PI-binding proteins, are directly involved in diseases such as cancer, diabetes and myopathies. Phosphatidylinositol-5-phosphate (PI-5-P) is the most recent PI to be identified;hence its function is poorly understood. PI-5-P has properties of a signaling molecule - its levels are low and can be rapidly regulated by extracellular cues. Remarkably, PI-5-P levels are up-regulated upon bacterial invasion of host cells, due to the activity of the bacterial virulence factors IpgD (from Shigella flexneri) and SigD/SopB (from Salmonella enterica), which are phosphatases that use PI-4,5-P2 as substrate. This pathological increase in PI-5-P levels is accompanied by dramatic changes in the actin cytoskeleton and in phosphoinositide 3-kinase (PI3k) signaling. On the other hand, PI-5-P levels can be down-regulated by PIP4k, an enzyme that converts PI-5-P into PI-4,5-P2 . The ultimate goal of this project is to understand the biological role of PI-5-P under physiological or pathological conditions. Our current hypothesis is that PI-5-P facilitates PI3k signaling by promoting local changes in the actin cytoskeleton. This hypothesis is based on evidence that both PI-5-P and PI-4,5-P2, together with the enzymes that catalyze the inter-conversion of these two lipids (PIP4k and IpgD), have been directly associated with actin rearrangements and PI3k signaling.
Our specific aims are: (i) To identify the enzymes that regulate basal or inducible levels of PI-5-P. Using HPLC analysis of metabolically labeled lipids, we will measure the temporal changes in PI-5-P levels caused by extracellular stimuli and how expression or knockdown of various lipid kinases or phosphatases can affect this lipid. Using cell fractionation, we will determine the subcellular compartments where PI-5-P is present. (ii) To characterize the effect of IpgD-induced PI-5-P on vesicle trafficking. We will express wild-type IpgD or membrane-targeted-IpgD to induce PI-5-P accumulation in specific membrane compartments. Using cell fractionation, we will determine the subcellular localization of IpgD-induced PI-5-P. Using live-cell microscopy, we will monitor the effect of IpgD expression in membrane structure and vesicle trafficking. (iii) To determine the role of PI-5-P in actin rearrangement and in PI-3,4,5-P3 internalization. The effect of PI-5-P on actin filament dynamics will be determined in a cell-free system, using fluorescence and TIRF microscopy. Using cells stably expressing GFP-actin, we will analyze the changes in actin dynamics in real-time at the specific sub-membrane compartments where PI-5-P is being generated. Using PI-3,4,5-P3- specific probes we will investigate the effect of PI-5-P in the internalization and degradation of PI-3,4,5-P3. Unraveling the molecular mechanisms for PI-5-P function in cells will contribute to the resources available for safely intervening in disease states associated with malfunctions in cell-to-cell signaling.

Public Health Relevance

Phosphoinositides are lipid molecules that play critical roles in cell-to-cell communication by working as intracellular messengers of hormones (e.g., insulin). In 1997, we discovered a new phosphoinositide in cells (PI-5-P) and, since then, we have found evidence that PI-5-P improves cellular response to insulin and promotes bacterial invasion.
Our aim i s to understand the function of this lipid under physiological and pathological conditions, with the goal of designing new strategies to prevent and treat diseases such as diabetes and bacterial infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK063219-10
Application #
8325148
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Silva, Corinne M
Project Start
2002-12-01
Project End
2013-02-16
Budget Start
2012-09-01
Budget End
2013-02-16
Support Year
10
Fiscal Year
2012
Total Cost
$228,316
Indirect Cost
$115,845
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472
Mackey, Ashley M; Sarkes, Deborah A; Bettencourt, Ian et al. (2014) PIP4k? is a substrate for mTORC1 that maintains basal mTORC1 signaling during starvation. Sci Signal 7:ra104
Sarkes, Deborah; Rameh, Lucia E (2010) A novel HPLC-based approach makes possible the spatial characterization of cellular PtdIns5P and other phosphoinositides. Biochem J 428:375-84