Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation

Abstract

PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380 -385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-crosslinking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains. PTEN3 is a phosphatidylinositol 3,4,5-triphosphate (PIP3) lipid phosphatase that is frequently inactivated in cancer by mutation, epigenetic silencing, or post-translational modifications (PTMs) (1-8). Loss of PTEN function allows unabated production of PIP3 from PIP2 by PI3Ks and stimulates the AKT protein kinase signaling pathway and cancer cell proliferation (1, 9, 10). Understanding the mechanisms of PTEN regulation is therefore critical to the development of therapeutics that can treat a wide variety of cancers.PTEN is a ϳ45-kDa protein composed of an N-terminal PTP-type phosphatase domain, a midsection phospholipid membrane interacting C2 domain, and a 50-residue regulatory tail (1). A crystal structure of the core PTEN catalytic-C2 region has revealed intimate interactions between the catalytic and C2 domains but omitted the C-terminal tail, which is considered to be flexible and largely unstructured (11). One of the most intensively studied set of PTMs in PTEN is a cluster of four C-terminal phosphorylations at Ser 380 , Thr 382 , Thr 383 , and Ser 385 (12-15). These phosphorylation events have been suggested to be catalyzed by protein kinases casein kinase 2 and/or glycogen synthase kinase 3␤ and demonstrated to drive a conformational change that inhibits membrane interactions and reduces catalytic activity (12-15).Several reports that shed light on the molecular basis for this phospho-dependent regulatory event suggest that the PTEN tail phosphorylation induces conformational closure involving intramolecular interactions between the tail and the CBR3 loop of the C2 domain of PTEN (14 -16). H...

Analysis of Site‐Specific Phosphorylation of PTEN by Using Enzyme‐Catalyzed Expressed Protein Ligation

“…In the PTEN enzymatic assays, all four monophosphorylated Y‐PTEN forms showed a decrease in catalytic activity relative to truncated recombinant PTEN (t‐PTEN, aa1–378) or non‐phosphorylated full‐length semisynthetic PTEN (n‐Y‐PTEN; Figure B and Table ). pSer380‐Y‐PTEN showed a threefold reduction in k cat / K M relative to Y‐n‐PTEN, which is similar to what had been seen previously with pSer380‐C‐PTEN . However, pThr382‐, pThr383‐, and pSer385‐Y‐PTENs each showed a six‐ to ninefold catalytic reduction compared with n‐Y‐PTEN, which are markedly greater declines than the two‐ to threefold inhibition seen with the corresponding monophosphorylated C‐PTENs .…”

“…The Y‐PTENs were analyzed by western blot by using a commercially available polyclonal anti‐phospho‐PTEN antibody that was previously generated with a triphosphorylated‐pSer380, pThr382, pThr383‐peptide (Figure A). It was previously reported with phosphorylated Cys379‐PTEN (C‐PTEN) forms that only pSer380 was detected over background with this antibody . In contrast, the pSer380, pThr383, and pSer385 Y‐PTEN forms were each readily illuminated with the anti‐phospho‐PTEN antibody, whereas pThr382‐Y‐PTEN was the only monophosphorylated Y‐PTEN that showed a background signal with this antibody (Figure A).…”

“…Analysis of this PTEN showed that it adopted a closed conformation and was less active toward PIP 3 to a much greater degree than was seen with C379 4p PTEN . This difference between C379 and Y379 4p‐PTENs suggests that previous studies with monophosphorylated (1p) PTENs, which incorporated the Y379C mutation, may also not fully capture the effect of tail phosphorylation on PTEN. To more accurately understand the effect of tail monophosphorylation events on PTEN activity and structure, we again exploited enzyme‐catalyzed EPL to create each of the wild‐type semisynthetic monophosphorylated PTENs (Scheme ), and below we describe their production and the analysis of their enzymatic properties.…”

“…Classical EPL involves a chemoselective ligation of an N ‐Cys synthetic peptide with a recombinant protein thioester generated by intein rearrangement. Biochemical and biophysical analyses of the semisynthetic phosphorylated and non‐phosphorylated PTENs revealed that C‐tail phosphorylation drives a “closed” conformation, in which the phosphorylated tail interacts with the C2 and phosphatase domains of PTEN and weakens phospholipid interactions . These semisynthetic PTENs all contained a Y379C mutation to facilitate the native chemical ligation reaction required for EPL, but it was later found that this mutation is linked to anomalous PTEN membrane association in vivo …”

Enzyme-catalyzed expressed protein ligation

“…Fraction phosphorylated: Untreated, 0.72 ± 0.06; Treated, 0.33 ± 0.09 (n = 5 biological replicates, p = 0.0071, Student's t-test). Note that it has previously been shown that phosphorylation can stabilize cellular PTEN 20 , explaining the reduction in total PTEN after CK2 inhibition. ( b ) Activity of t-, Y379-n-, C379-4p-, and Y379-4p-PTEN toward 160 μM soluble di-C6 PIP 3 with 60 mM or 200 mM NaCl.…”