Novel Homeostatic Mechanism Tunes PI(4,5)P2-dependent Signaling at the PM

Posted on July 20, 2022

Website Rachel Wills Research Spotlight

Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) controls many aspects of plasma membrane functions, and is thus essential for life in mammals. Some of these functions include the selective permeability of the plasma membrane, attachment to the cytoskeleton, regulation of proteinaceous cargo transport, extracellular signaling, and facilitation of bacterial and viral pathogens.

Genetic defects of PI(4,5)P2 synthesis that occur either increase or decrease the production of PI(4,5)P2. These defects lead to diseases such as cancer, kidney disease, and dysentery. It is obvious that strict regulation of PI(4,5)P2 synthesis is necessary for healthy plasma membrane function, but what is not obvious is the mechanism by which PI(4,5)P2 levels are regulated.

Past work has centered around the idea that phosphatidylinositol 4-phosphate 5-kinases (PIP5K) enzymes are positively regulated. However, Dr. Gerry Hammond and his group at the University of Pittsburgh, which includes Rachel Wills, hypothesized that negative feedback actually maintains tonic levels of PI(4,5)P2.

Testing the Hypothesis

To better understand the mechanism by which PI(4,5)P2 is regulated, Dr. Hammond’s group initially overexpressed all isoforms of human PIP5K (A-C) and then measured PI(4,5)P2 levels. The overexpression of PIP5K led to substantial increase in PI(4,5)P2 levels in all cases. They then overexpressed phosphatidylinositol 5-phosphate 4-kinases (PIP4Ks) which produce PI(4,5)P2 albeit in much lesser quantities than PIP5Ks. Overexpression of PIP4Ks actually led to a slight decrease in PI(4,5)P2 levels. PIP4K overexpression was able to attenuate the increased PI(4,5)P2 levels caused by overexpression of PIP5Ks.

They then went on to hypothesize that PIP5K inhibition by PIP4K actually requires the substrate itself, PI(4,5)P2. Through a series of experiments their data supported the hypothesis and found that this mechanism is dependent on PI(4,5)P2. They also tested whether PI(4,5)P2 enhances the recruitment of PIP4K2C, the most abundant PIP4K in HeLa cells. They showed that PIP4K2C binds PI(4,5)P2 with low affinity in the plasma membrane and that PIP4Ks are low-affinity effectors of PI(4,5)P2 capable of sensing both a decrease and increase in PI(4,5)P2 levels at the plasma membrane.

All of these observations were consistent with a direct binding interaction between PIP5K and PIP4K, but could also have indicated a PI(4,5)P2 induced co-enrichment on membranes. Using a bait protein, they were able to show that it is indeed a direct binding interaction between PIP5K and PIP4K rather than a PI(4,5)P2 induced co-enrichment.

Thus, they proposed the following homeostatic mechanism:

  • When PI(4,5)P2 levels rise due to PIP5K activity, PIP4K is recruited to the plasma membrane.
  • Here, PIP4K can bind and inhibit PIP5K.
  • If PI(4,5)P2 levels fall, PIP4K is released and results in disinhibition of PIP5K, as well as recovery of PI(4,5)P2

Understanding this Mechanism Makes Future Therapeutics for PI(4,5)P2 Synthesis Defects a Real Possibility

Understanding the mechanism by which PI(4,5)P2 levels are regulated in the mammalian plasma membrane allows the possibility of determining which PI(4,5)P2 dependent functions are dysregulated by different pathological conditions. Being able to determine these dysregulations brings us closer to finding potential therapeutic targets for these conditions.

Thank you, Rachel Wills and Dr. Gerry Hammond, for allowing us to share this incredible research!

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