Subcellular localization and functional analysis of tumor-associated N-terminal PTEN mutations.

<p>(<b>A</b>) Amino acid sequence of PTEN N-terminus (residues 1–43). The nuclear localization signal (NLS), the PIP2-binding motif (PBM), and the cytoplasmic localization signal (CLS) are indicated. The KRR motif (residues 13–15) is in red. (<b>B, C</b>) The influence of tumor-associated PTEN mutations on PTEN subcellular localization was assessed by immunofluoresecence on mammalian COS-7 cells. In B, COS-7 cells transfected with wild type (wt; 1–403) or mutated PTEN (1–375, or mutations in a 1–375 background) were analysed by standard immunofluorescence microscopy using anti-PTEN 425A mAb (green). Nuclei were stained with Hoechst (blue). In C, the nuclear/cytoplasmic distribution of PTEN mutations was scored from COS-7 cells processed as in B; N, nuclear localization; C, cytoplasmic localization; N/C, nuclear/cytoplasmic localization. (<b>D, E</b>). The influence of tumor-associated PTEN mutations in the <i>in vivo</i> PTEN PIP3 phosphatase activity was assessed in yeast. In D, <i>S</i>. <i>cerevisiae</i> drop growth experiments were performed on co-transformants expressing p110α-CAAX and the indicated PTEN mutations, under repression (Glucose) or induction (Galactose) of the expression of the heterologous mammalian proteins. Expression of p110α-CAAX results in high toxicity in yeast cells as a result of the depletion of essential PIP2, which is converted to PIP3. Co-expression of wild type PTEN hydrolyses PIP3 and restores PIP2 levels and viability. In E, PIP3 hydrolysis by PTEN in yeast cells was monitored indirectly using a GFP-Akt1 reporter protein. Results are shown as percentage of yeast cells that display GFP-Akt1 at the plasma membrane (PM). The bottom panel shows the equivalent expression in the yeast of all PTEN mutations, as assessed by immunoblot using anti-PTEN and anti-actin (as a loading control) antibodies.