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-cross-linking 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.

Keywords: phosphatase; phosphatase and tensin homolog (PTEN); phosphorylation; post-translational modification (PTM); protein conformation; pten.

FIGURE 1.

Generation of unmodified, mono-, di-, tri-, and tetraphosphorylated semisynthetic PTEN proteins. A, PTEN is composed of a protein-tyrosine phosphatase (PTPase) domain, a C2 domain, and a regulatory C-terminal tail. The cluster of phosphorylation (Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵) is highlighted. B, C-terminal t-PTEN (amino acids 1–378) with a thioester at the C terminus is generated from intein fusion, treated with MESNA, and then ligated to the synthetic peptide containing different combinations of Ser(P) and Thr(P) (mono: p380, p382, p383, and p385; di: 2p-380/382, 2p-380/385, and 2p-382/383; tri: 3p-380/382/383; and tetra: 4p-380/382/383/385). X, Ser/Thr/Ser(P)/Thr(P). C, Coomassie-stained 10% SDS-PAGE gel of the set of differentially phosphorylated semisynthetic PTEN proteins. Ligation of t-PTEN-thioester and the specific peptide proceeds at a constant rate for 48 h, and the full-length PTEN is further purified by FPLC-anion exchange chromatography using MonoQ column. The final protein is >90% pure. First lane, n-PTEN; second lane, p380-PTEN; third lane, p382-PTEN; fourth lane, p383-PTEN; fifth lane, p385-PTEN; sixth lane, 2p-380/382-PTEN; seventh lane, 2p-382/383-PTEN; eighth lane, 2p-380/385-PTEN; ninth lane, 3p-380/382/383-PTEN; tenth lane, 4p-PTEN.

FIGURE 2.

Catalytic activity of the set of differentially phosphorylated semisynthetic PTENs toward a range of diC6-PIP3 substrate concentrations. A, n-PTEN and monophosphorylated PTEN kinetics. B, diphosphorylated PTEN kinetics. C, tri- and tetraphosphorylated PTEN kinetics (n = 2).

FIGURE 3.

Alkaline phosphatase sensitivity of the set of differentially phosphorylated semisynthetic PTENs. A, Western blot of the set of differentially phosphorylated semisynthetic PTENs using an anti-phospho-PTEN (Ser³⁸⁰, Thr³⁸², and Thr³⁸³) antibody. First lane, n-PTEN; second lane, p380-PTEN; third lane, p382-PTEN; fourth lane, p383-PTEN; fifth lane, p385-PTEN; sixth lane, 2p-380/382-PTEN; seventh lane, 2p-382/383-PTEN; eighth lane, 2p380/385-PTEN; ninth lane, 3p-380/382/383-PTEN; tenth lane, 4p-PTEN. B, time course of the rate of dephosphorylation of the p380-containing semisynthetic PTENs after alkaline phosphatase treatment analyzed by Western blotting. C, quantification of the time courses of the Western blot data in Fig. 3B (n = 2). Error bars indicate ± standard error.

FIGURE 4.

SDS-PAGE analysis and catalytic activity of a series of mutant semisynthetic PTENs toward a range of diC6-PIP3 substrate concentrations. A, Coomassie-stained 10% SDS-PAGE gel of semisynthetic PTEN mutants. First lane, molecular mass markers; second lane, BB-4p-PTEN; third lane, Cα2A-4p-PTEN; fourth lane, Cα2A-t-PTEN; fifth lane, Cα2D-4p-PTEN; sixth lane, Cα2D-t-PTEN; seventh lane, 4p-ePTEN; eighth lane, 3R/D-4p-PTEN; ninth lane, 3R/D-t-PTEN. B, catalytic activity of ePTEN forms with diC6-PIP3 (n = 2): Q17R, R41G, E73D, N262Y, and N329H. C, catalytic activity of Cα2 loop mutant PTEN forms with diC6-PIP3. Cα2D, K327D, K330D, K332D, and R335D; Cα2A, K327A, K330A, K332A, and R335A (n = 2).

FIGURE 5.

Alkaline phosphatase sensitivity of different semisynthetic phospho-PTEN mutants. A, Western blot of time course from the alkaline phosphatase-treated 4p-ePTEN (Q17R, R41G, E73D, N262Y, and N329H) and WT 4p-PTEN. B, quantification of the alkaline phosphatase assay for 4p-ePTEN and WT 4pPTEN (n = 3). C, Western blot of time course from the alkaline phosphatase treated Cα2D-4p-PTEN (K327D, K330D, K332D, and R335D), Cα2A-4p-PTEN (K327A, K330A, K332A, R335A), and WT 4p-PTEN. D, quantification of the alkaline phosphatase assay for 4p-Cα2 loop mutants and WT 4p-PTEN (n = 6). Error bars indicate ± standard error.

FIGURE 6.

BB-4p-PTEN, photo-cross-linking, and mass spectrometry analysis. A, MALDI-TOF MS spectrum of 18-mer synthetic peptide containing tetraphosphorylation (Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵), Bpa at position 392, and a C-terminal biotinylated lysine used for expressed protein ligation. B, isotopic distribution of a 4+ charged cross-linked peptide with the reported mass, m/z, and Δm from Crossfinder. C, high resolution tandem-MS spectrum of the cross-linked peptide. D, PTEN crystal structure (Protein Data Bank code 1D5R) highlighting the site of cross-linking and other important areas shown to be involved in phospho-C-tail binding.

FIGURE 7.

Conformational analysis of the 3R/D PTEN mutant. A, catalytic activity of WT t-PTEN (123 ± 4 nmol/min/mg) compared with 3R/D t-PTEN (<4 nmol/min/mg) with 160 μm diC6-PIP3 (n = 2). B, Western blot of time course of dephosphorylation of alkaline phosphatase-treated 3R/D-4p-PTEN (R41D, R47D, and R74D) and WT 4p-PTEN. C, quantification of the alkaline phosphatase assay for 3R/D-4p-PTEN and WT 4p-PTEN (n = 6). Error bars indicate ± standard error.