Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Tobias Baumgart

Abstract

Phosphoinositides, though rare species in cell membranes, play important roles in cell regulation and signal transduction mediation. Two vital phosphoinositides are PI(4,5)P2 and PI(3,4,5)P3, which are interconverted by a kinase, PI3K, and a phosphatase, PTEN. PI(3,4,5)P3 acts as a second messenger to recruit and activate Akt to trigger downstream signaling pathways for cell proliferation and growth. Regulation of PI(3,4,5)P3 amounts on cell membranes is critical, however, detailed regulatory mechanisms are not well known yet. First, the mechanism of PI(3,4,5)P3 hydrolysis by PTEN is still under debate. Second, how Ras GTPase affects PTEN-mediated PI(3,4,5)P3 degradation is not well known. Third, the bistability of PI(3,4,5)P3 and PI(4,5)P2 has been hypothesized to be important in maintaining cell polarity and the cell fate switch, but how this bistability is achieved by the collaboration of PTEN / PI3K is also unknown.

To answer the above questions, first we studied the binding affinity and specificity of sensor proteins to the phosphoinositides. YFP-PH-Grp1 and mCherry-PH-Grp1 are shown to bind PI(3,4,5)P3 specifically, while EGFP-PH-PLCδ1 is shown to bind PI(4,5)P2 specifically. Fluorescent protein-labeled Grp1 and PLCδ1 are then used to quantify and monitor PI(3,4,5)P3 and PI(4,5)P2 amounts in the presence of enzymes by either TIRF or confocal microscopy.

Second, we characterized PTEN-mediated PI(3,4,5)P3 dephosphorylation and PI3K-mediated PI(4,5)P2 phosphorylation reactions on model lipid membranes. The rate of PI(3,4,5)P3 hydrolysis by PTEN increases with increasing PI(4,5)P2 concentration, suggesting product-mediated positive feedback loop. By fitting kinetic traces of PI(3,4,5)P3 hydrolysis with the theoretical model we developed, the origin of this feedback was proposed to arise from membrane recruitment and product-mediated activation of PTEN. PI(4,5)P2 phosphorylation by PI3K, which is activated by phosphopeptide binding, is shown to follow typical Michaelis-Menten kinetics. Membrane-bounds KRas is shown to activate PI3K synergistically with phosphopeptide and is shown to slow down PI(3,4,5)P3 hydrolysis by PTEN.

Finally, we combined both PTEN and PI3K on bead-supported lipid bilayers and observed that the steady-state of PI(4,5)P2 / PI(3,4,5)P3 distribution depends not only on the PTEN / PI3K ratio, but also on the initial state of phosphoinositides. This phosphorylation-dephosphorylation cycle is shown on model lipid membranes to exhibit hysteresis, which is the hallmark of bistability. By kinetic model simulation, we identified that the PTEN-PI(4,5)P2 positive feedback loop is critical in establishing phosphoinositide bistability. This bistability can also be affected by the presence of sensor proteins due to competitive substrate binding, leading to the bimodal distribution of PI(4,5)P2-rich and PI(3,4,5)P3-rich states at an intermediate PTEN / PI3K ratio.

Embargoed

Available to all on Sunday, July 03, 2022

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Included in

Chemistry Commons

Share

COinS