Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery and functional characterization of a neomorphic PTEN mutation.

doi: 10.1073/pnas.1422504112

Figure Lengend Snippet: Fig. 1. Summary of PTEN amino acid 126. (A) PTEN domains and catalytic site (P-loop) conservation across species. PTEN is a 403-amino acid protein with five functional domains. The catalytic “P-loop” re- gion (yellow) spans amino acids 123–130 and is completely conserved across a broad range of eu- karyotes. The catalytic cysteine residue at position 124 is highlighted in purple, and amino acid 126 is highlighted in green. (B) PTEN protein structure with catalytic P-loop, residue 124, and residue 126 high- lighted in yellow, purple and green, respectively. (C) Amino acid comparison of the catalytic CX5R motif between WT PTEN, PTENA126G, and Ci-VSP. Gray boxes represent conserved regions. (D) PTEN WT (red) and p.A126G (gray) binding pocket over- lay. (E) Binding free energies of 5- and 3-oriented Ins(1,3,4,5)P4 to different PTEN alleles. Arrows in- dicate the direction of ΔΔG5→3

Article Snippet: PTEN WT (Addgene plasmid 10750), p.C124S catalytic dead (Addgene plasmid 10744), and p.A126G mutant expression vectors were used for transient transfections into the PC-3 cells.

Techniques: Functional Assay, Residue, Comparison, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery and functional characterization of a neomorphic PTEN mutation.

doi: 10.1073/pnas.1422504112

Figure Lengend Snippet: Fig. 3. In vitro characterization of PTEN amino acid 126. (A and B) Phospholipids were isolated from cell lysates in cells expressing PTEN-WT, PTEN-C124S, or PTEN- A126G. PIP3 and PI(3,4)P2 levels were quantified by ELISA, and relative abundance was compared with catalytic dead p.C124S expression (n = 3). (A) There is no statistical significance to the observed difference in PIP3 between mutant p.A126G and WT alleles (N.S.); however, differences between mutant p.A126G and catalytic dead p.C124S PIP3 levels are significant (*P ≤0.05). (B) Differences in PI(3,4)P2 levels between mutant p.A126G and catalytic dead p.C124S alleles are statistically significant (*P ≤0.05) as are differences between mutant p.A126G and WT alleles (*P ≤0.05). (C) Activation of PI3K/Akt pathway in PC-3 cells. Total cell lysates of PC-3 cells transfected with PTEN alleles and drug treated with PI3K-p110α inhibitor (BYL719) or PI3K-p110β inhibitor (AZD6482) were resolved by SDS/PAGE and probed with the indicated antibodies. (D) Phospho-Akt levels were normalized over total protein levels and relative abundance was compared with WT expression (n = 4). We observed no statistically significant difference between mutant p.A126G and catalytic dead p.C124S mutations (N.S.) but did observe a significant difference between WT and p.A126G protein levels (*P ≤0.05). (E) Phospho-S6 levels were normalized over total protein levels, and relative abundance was compared with WT expression (n = 4). There is no statistical significance to the difference between mutant p.A126G and catalytic dead p.C124S mutations (N.S.), but the difference between WT and p.A126G protein levels is significant at the *P ≤0.05 level. (F) Wound closure was quantified at 0 h, 12 h, and 24 h and normalized relative to 0-h levels using TScratch (n = 6). There is a statistical significance between mutant p.A126G and catalytic dead p.C124S at 24 h (*P ≤0.05). (G) Assessment of cell migration across PTEN alleles in the presence or absence of the PI3K inhibitor. Representative photographs were taken at 0 h, 12 h, and 24 h postwound. (Scale bar: 200 μm.) Error bars represent SD.

Article Snippet: PTEN WT (Addgene plasmid 10750), p.C124S catalytic dead (Addgene plasmid 10744), and p.A126G mutant expression vectors were used for transient transfections into the PC-3 cells.

Techniques: In Vitro, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Mutagenesis, Activation Assay, Transfection, SDS Page, Migration