Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis

Abstract

The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380–385) and these phosphorylations are proposed to induce a reduction in PTEN’s plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN’s regulation and suggest pharmacologic approaches for direct PTEN activation.DOI: http://dx.doi.org/10.7554/eLife.00691.001

Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

“…5A). To verify this, we used limited proteolysis, which is a wellcharacterized technique used to measure changes in the conformational stability and flexibility of proteins (54). Digestion of the purified ectodomain of nicastrin with varying amounts of trypsin in the presence or absence of reducing reagent revealed that reduced nicastrin was substantially more sensitive to proteolysis than the unreduced protein (Fig.…”

The functions of tumor suppressor PTEN in innate and adaptive immunity

“…15 In addition, binding of PTEN with the plasma membrane is also regulated by the phosphorylation status of PTEN. [16][17][18] This interaction is delineated in more detail in the following sections.…”

“…33 In addition, Tyr240 and Tyr315 phosphorylation could help to maintain phosphatase function of PTEN. 34,35 Bolduc et al 16 found that upon phosphorylation of the 380-385 Ser/Thr cluster, the PTEN C-terminal modified tail clamps down intramolecularly on the C2 domain, preventing PTEN from binding to the plasma membrane and reducing its catalytic action toward PIP3. Nguyen also proved that the phosphatase catalytic site forms the membrane-binding regulatory interface and interacts with the inhibitory phosphorylated C-terminal tail.…”

A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells

“…A cluster of serine and threonine residues (Ser380, Thr382, Thr383 and Ser385) in the regulatory C-terminal tail of PTEN is constitutively phosphorylated by casein kinase 2 (CK2). This results in a stable 'closed' form that has reduced lipid phosphatase activity, decreased plasma membrane targeting and increased conformational compaction [15][16][17][18][19] . Intramolecular interaction of the phosphorylated C-terminal tail with basic residues within the N-terminal PIP2-binding motif, the catalytic and C2 domains, maintains PTEN in its 'closed' form 20,21 .…”

“…Mutation of the C-terminal tail phosphorylation cluster disrupts the intramolecular interaction and promotes an 'open' form of PTEN with increased catalytic activity and plasma membrane targeting. These findings have led to an elegant model whereby a phosphorylation-regulated switch controls the transition of PTEN from a 'closed' cytoplasmic inactive state and an 'open' active state that is localized to the plasma membrane 17,19,21 . PTEN is also phosphorylated in its C2 domain by ROCK, the downstream effector of RhoA, resulting in increased PTEN catalytic activity 22 .…”