The structural basis of PTEN regulation by multi-site phosphorylation

PTEN is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN’s catalytic activity, cellular membrane localization, and stability are orchestrated by a cluster of C-terminal phosphorylation events on Ser380, Thr382, Thr383, and Ser385 but the molecular details for this multifaceted regulation have been uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography, and computational simulations to obtain a detailed picture of how the phospho-C-tail interacts intramolecularly with PTEN’s C2 and phosphatase domains. A key element of this investigation involved characterizing the sea squirt homolog of PTEN, the voltage-sensing phosphatase (VSP), which was amenable to NMR mapping of the interactions of individual tail phospho-sites with particular VSP amino acid residues. What emerged from this multi-disciplinary structural analysis is an extended phosphorylated C-tail peptide that belts around much of VSP/PTEN and coats the phospholipid membrane binding interface and protrudes into the enzyme active site. This structural model provides a framework for how C-tail phosphorylation can comprehensively impact PTEN’s cellular functions.