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

The lipid molecule phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) controls virtually all aspects of plasma membrane (PM) function in animal cells. These functions range from cellular signaling to selective membrane permeability to the attachment of the cytoskeleton. Upon the reduction of PI(4,5)P2 these cellular functions are dysregulated. The majority of PI(4,5)P2 is synthesized by type 1 phosphatidylinositol 4-phosphate 5-kinase (PIP5K). However, the mechanism by which PIP5K localizes to the PM to access its substrate phosphoinositide 4-phosphate (PI4P) is not well known. Therefore, understanding the regulation of this kinase is critical for determining how the cell maintains PI(4,5)P2 homeostasis. We observed PM localization of over-expressed PIP5K isoforms and endogenously tagged PIP5K1A to determine the potential mechanisms for PM association. After experimentally altering the phosphoinositide composition of the PM, we were able to observe the acute corresponding changes in PIP5K localization. PIP5K remained localized with the PM in the presence of PI4P and PI(4,5)P2. Furthermore, we determined that two basic residues in the C-terminal tail are necessary for this PM interaction. The crystal structure of PIP5K displays these two residues opposite the activation loop and near an unstructured insert loop. Ultimately, we believe that the insert loop functions as a PI(4,5)P2 binding pocket or confers allosteric regulation to reveal a cryptic PI(4,5)P2 binding site on PIP5K to facilitate its PM localization. On the other hand, increased levels of PI(4,5)P2 are also associated with a wide range of diseases. Yet, it remains unclear how cells sense and maintain PI(4,5)P2 levels without exceeding the basal level. Here, we show that the type 2 phosphatidylinositol 5-phosphate 4-kinase (PIP4K) family of enzymes, that synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. We show that PIP4K inhibits the synthesis of PI(4,5)P2 and that this is done through a direct interaction with PIP5K. Furthermore, PIP4K is recruited to the membrane to interact with PIP5K in a PI(4,5)P2 dependent manner. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P¬2-dependent signaling to elevated PI(4,5)P2 levels. We find that a subset of PI(4,5)P2 mediated functions may drive diseases associated with disrupted PI(4,5)P2 homeostasis