A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN

Abstract

The PI 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), one of the most important tumor suppressors, must associate with the plasma membrane to maintain appropriate steady-state levels of phosphatidylinositol 3,4,5-triphosphate. Yet the mechanism of membrane binding has received little attention and the key determinants that regulate localization, a phosphatidylinositol 4,5-bisphosphate (PIP2) binding motif and a cluster of phosphorylated C-terminal residues, were not included in the crystal structure. We report that membrane binding requires PIP2 and show that phosphorylation regulates an intramolecular interaction. A truncated version of the enzyme, PTEN1-351, bound strongly to the membrane, an effect that was reversed by co-expression of the remainder of the molecule, PTEN352-403. The separate fragments associated in vitro, an interaction dependent on phosphorylation of the C-terminal cluster, a portion of the PIP2 binding motif, integrity of the phosphatase domain, and the CBR3 loop. Our investigation provides direct evidence for a model in which PTEN switches between open and closed states and phosphorylation favors the closed conformation, thereby regulating localization and function. Small molecules targeting these interactions could potentially serve as therapeutic agents in antagonizing Ras or PI3K-driven tumors. The study also stresses the importance of determining the structure of the native enzyme.iRAP ͉ phosphatase ͉ PI3K ͉ PIP2 ͉ PIP3 P TEN (phosphatase and tensin homologue deleted on chromosome 10) is a lipid phosphatase that dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PIP 3 ) and opposes PI3K signaling (1). This important tumor suppressor is frequently mutated in endometrium, prostate, and brain cancer, and alterations of PTEN activity leads to Cowden and Bannayan-Zonana syndromes in which patients develop benign hamartomas (2-4). Mice homozygous for a deletion of PTEN die in early embryogenesis whereas heterozygotes have a propensity to develop widespread neoplasms (5). Prostate-directed conditional deletions of PTEN result in metastatic prostate cancer (6-8). Neural and astrocyte-specific deletions lead to greatly enlarged brain size and abnormalities in astrocytes and neurons (9). Cultured mammalian cells lacking PTEN proliferate faster, resist apoptosis, and migrate aberrantly (10-12). Dictyostelium discoideum cells lacking PTEN have elevated PIP 3 and are severely defective in chemotaxis (13).Regulation of PTEN activity, localization, and function are controlled by a variety of mechanisms. NOTCH1, by acting through transcription factors CBF-1 and MYC or HES-1, respectively, has been reported to either increase or decrease PTEN expression (14-16). Another level of regulation is post-translational modification. Acetylation by p300/CBP-associated factor blocks PTEN activity (17). PTEN catalytic activity is negatively regulated by reactive oxygen species under oxidative stress (18-20) by disulfide linkage of catalytic cysteine 124 with cysteine 71 (18...

SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane

“…In fact, PTEN has also been found to directly translocate to the plasma membrane in certain cell lines and under specifi c conditions 3,20,31 . Our fi nding that SUMO1 modifi cation of PTEN increases PTEN binding to the plasma membrane ( Figs 4, 5 and 7 ) may explain why a small fraction of PTEN acts through the dynamic interaction with the inner face of plasma membrane 2 .…”

“…It has been proposed in the PTEN open / closed model that phosphorylation at S 370 / S 380 / T 382 / T 383 / S 385 of the C-terminal tail regulate membrane association 3,33 . Th is model is also involved in binding of the C2 domain to phosphatidylserine 4 and the PIP2-binding motif to PIP2 7,34 .…”

“…PTEN is mainly found in the cytosol and nucleus. Several studies have shown that under normal conditions, only a small fraction of cytosolic PTEN is dynamically bound to the plasma membrane, because PTEN in a constrained conformation has a low affi nity for membrane binding 2,3 . It is still poorly understood how cytosolic PTEN interacts with its main substrate PIP3 localized at the inner face of plasma membrane, although several elements / factors, including the CBR3 loop in the C2 domain 4 -6 , N-terminal PIP2-binding motif ( Fig.…”

“…It is still poorly understood how cytosolic PTEN interacts with its main substrate PIP3 localized at the inner face of plasma membrane, although several elements / factors, including the CBR3 loop in the C2 domain 4 -6 , N-terminal PIP2-binding motif ( Fig. 1a ) and PIP2 6,7 , C-terminal tail phosphorylations 2,3,8 -10 and integrity of the phosphatase 2,3 , have been suggested to be involved in the membrane binding and activation of PTEN.…”

Targeting the translational apparatus to improve leukemia therapy: roles of the PI3K/PTEN/Akt/mTOR pathway

“…These phosphorylated residues serve to lock PTEN in a stable closed conformation, which reduces both its membrane localization and lipid phosphatase activity. 57,58 There are multiple kinases that phosphorylate PTEN, including casein kinase 2, and GSK-3b, which may occur at other residues including T366 and S370. 55,59,60 Proteins such as PICT1 and RAK were initially identified as proteins that bind PTEN.…”