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

<p>(<b>A</b>) Nuclear/cytoplasmic distribution of PTEN N-terminal mutations, monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119287#pone.0119287.g002" target="_blank">Fig 2C</a>. Nuclear/cytoplasmic distribution of mutations V9A to E18A is as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119287#pone.0119287.ref018" target="_blank">18</a>]. (<b>B</b>) Influence of PTEN N-terminal mutations in the <i>in vivo</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119287#pone.0119287.g002" target="_blank">Fig 2E</a>. In the middle panel (drop growth), growth was monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119287#pone.0119287.g002" target="_blank">Fig 2D</a>. The activity of mutations K6A to E18A is as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119287#pone.0119287.ref036" target="_blank">36</a>]. In the bottom panel, the equivalent expression in the yeast of all PTEN mutations, as assessed by immunoblot using anti-PTEN antibodies, is shown. <b>(C)</b> 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 <i>in vivo</i> 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.