Subcellular localization and functional analysis by Ala-scanning of PTEN N-terminal region reveals distinct subgroups of PTEN mutations.

(A) Nuclear/cytoplasmic distribution of PTEN N-terminal mutations, monitored as in Fig 2C. Nuclear/cytoplasmic distribution of mutations V9A to E18A is as in [18]. (B) Influence of PTEN N-terminal mutations in the in vivo PTEN PIP3 phosphatase activity, assessed in yeast. In the upper panel (bars graph), the PIP3 phosphatase activity of PTEN N-terminal mutations was monitored as in Fig 2E. In the middle panel (drop growth), growth was monitored as in Fig 2D. The activity of mutations K6A to E18A is as in [36]. In the bottom panel, the equivalent expression in the yeast of all PTEN mutations, as assessed by immunoblot using anti-PTEN antibodies, is shown. (C) Scheme summarizing the Ala-scanning functional results and the distinct subgroups of PTEN mutations. PTEN N-terminal amino acid sequence (residues 1–43) is shown, and the NLS (grey) motif is indicated. The functional consequences of each Ala-substitution are indicated with a colour code: green (+ +), no effect; yellow (- +), normal PIP3 phosphatase activity but impaired nuclear accumulation; blue (+-), impaired phosphatase activity but normal nuclear accumulation; red (—), impaired phosphatase activity and nuclear accumulation. For nuclear localization in the background of PTEN 1–375, we consider positive those mutants that showed over 50% of cells with nuclear localization. For in vivo activity on PIP3, we consider positive those mutants that showed under 30% of cells with Akt at the plasma membrane and significantly rescued p110α-induced growth inhibition. The first Met (M) was not mutated, and corresponds to PTEN wild type.