Fig. 2.

Genetic targeting of PTEN to membrane rafts abolishes PDK1 activation, Akt membrane recruitment, and Akt activity. (A) PTEN is primarily localized to nonraft regions of the plasma membrane. Crude plasma membranes from HEK 293 cells were solubilized and subjected to sucrose density gradient fractionation, followed by Western blotting with an anti-PTEN antibody. CTB was used as a raft marker. Anti-tubulin was used to ensure the separation of membrane proteins from cytosolic proteins. (B) Statistically significant differences between PTEN levels in rafts and nonraft regions (***P < 0.001; n = 3). Densitometric analysis indicated that the majority of the membrane PTEN resides in nonraft regions. (C) PTEN A4, fused with a C-terminal fluorescent protein (FP), was targeted to membrane rafts and nonraft regions with a Lyn or Kras motif. (D) The membrane localization of Lyn-PTEN A4 or PTEN A4-Kras was verified by sucrose density gradient fractionation of total cell lysates of HEK 293 cells expressing Lyn-PTEN A4-YFP or PTEN A4-YFP-Kras. CTB was used as a raft marker. Targeted PTEN A4 was detected with an anti-GFP antibody. Yellow fluorescence images show the membrane localization of PTEN. (E) Representative time courses indicating that the response of Lyn-PARE was abolished by Lyn-PTEN A4 (n = 5; PTEN fused with mCherry), but not PTEN A4-Kras (n = 7). (F) Representative time courses demonstrating that the membrane translocation of the Akt PH domain was abolished by Lyn-PTEN A4 (n = 4; PTEN fused with mCherry), but not PTEN A4-Kras (n = 4). (G) Representative time courses indicating that the response of AktAR was abolished by Lyn-PTEN A4 (n = 7; PTEN fused with mCherry), but not PTEN A4-Kras (n = 6).