J. Biol. Chem. 290, 1592–1606 (2015)

PTEN is a lipid phosphatase that acts as a tumor suppressor through its action on the PI3K/Akt signaling pathway. PI(3,4,5)P2 (phosphatidylinositol 4,5 bisphosphate) is the most preferred PTEN substrate, but PI(3,4)P2 and PI(3)P can also be dephosphorylated by PTEN. Another PIP2, PI(4,5)P2, potentiates PTEN activity towards PI(3,4)P2 and PI(3)P by enhancing the enzyme's binding to membranes. PI(4,5)P2 kinetic activation studies, structural studies and molecular dynamics simulations have implicated an N-terminal PTEN loop as the PI(4,5)P2 binding site. Besides this site and the substrate-binding site, an amphiphile-binding site (known as AHS) defines a third functionally distinct region of the catalytic domain of PTEN. To locate more specifically the PI(4,5)P2 binding site and the regulation by PI(4,5)P2, Wei et al. characterized the binding and activity of a series of phosphatidylinositols to PTEN using high-resolution field cycling 31P NMR spectroscopy on spin-labeled protein along with enzyme kinetics. They found that PI(4,5)P2 differentially acts as an inhibitor or activator of PTEN depending on the substrate (PI(3)P or PI(3,4)P2, respectively). Mutation of Lys13, a residue important in PI(4,5)P2 activation of PTEN, or Arg47 in the AHS site abrogated the ability of PI(4,5)P2 to activate PTEN, suggesting that the PI(4,5)P2 activator site is near the AHS and is distinct from the AHS (which they also found to involve Arg47). The NMR experiments further validated three distinct sites, where PI(4,5)P2 is clearly not binding the active site, and showed that even when PI(4,5)P2 is unable to activate PTEN (when Lys13 or Arg47 is mutated), it does still bind the enzyme. The results further suggest that the orientation of PI(4,5)P2 within PTEN is very specific, as might be expected, as is its close proximity to the substrate lipid. The authors propose a model where PI(4,5)P2 enhances anchoring of PTEN to the membrane by limiting flexibility of the N-terminal PTEN domain and acts as an electrostatic anchor for processive catalysis where substrate and product can swiftly enter and exit the active site.