The PAR-aPKC system: lessons in polarity. that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 CPDA comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and CPDA pAkt were dependent on dynamin activity. These results spotlight a novel signaling function of apical endosomes in polarized cells. INTRODUCTION Atypical protein kinase C (aPKC, comprising PKC/ and PKC) is essential for polarization in epithelia and neurons and is conserved in the evolution of multicellular organisms (Suzuki and Ohno, 2006 ). It is a central component of the Par3-Par6-aPKC polarity complex (Wang and Margolis, 2007 ). In epithelial cells, it controls the assembly and localization of tight junctions (Suzuki assessments of pairs of means; *p 0.025 and **p 0.005 indicate the probability of Bglap random differences from the average value immediately above (n = 3). (D) Caco-2 cells were transduced with mock lentiviral particles (mock) or with particles expressing anti-PDK1 shRNA and selected in puromycin. Confluent, differentiated cells not exposed to cycloheximide (0 h) were used to assess the efficacy of the knockdown and to control for apoptosis with antiCcaspase 3 (casp3) antibody. A 2-h incubation in 20 mM H2O2 of mock cells served as a positive control for apoptosis. Cells were treated (+) or not (C) with 10 g/ml cycloheximide for indicated periods of time for up to 24 h. Total SDS extracts were analyzed by immunoblotting with the antibodies indicated around the left. (E) The values CPDA from bands in three impartial experiments as described in D were expressed as described in C and plotted as a function of time. (F) For coimmunoprecipitation experiments, Caco-2 cells were incubated or not (contr) with 10 CPDA g/ml cycloheximide overnight (cyclo). The Triton-soluble fraction was immunoprecipitated with rabbit polyclonal anti-PDK1 antibody (+) or with nonimmune IgG, and analyzed by immunoblot for PDK1 or PKC. The same blot analysis was performed for samples of the supernatant after the immunoprecipitation. (G) Relative amount of PKC immunoprecipitated with PDK1 was calculated by normalizing the PKC signal to the PDK1 signal in the same immunoprecipitates. Data represent the mean SD from three impartial experiments. The averages of PKC immunoprecipitated in the presence or absence of cycloheximide were not significantly different. To ensure that the destabilization of PKC was PDK1 specific, we knocked down this protein with short hairpin RNA (shRNA) delivered by lentivirus particles. The efficiency of the knockdown estimated by immunoblot was approximately 87% (Physique 1D). Of importance, although the PDK1-knockdown cells grew at a much slower rate than the mock-infected controls, we could not detect apoptosis by caspase 3 cleavage (Physique 1D). We performed a 24-h time course after addition of cycloheximide. Once again, mock-transduced cells showed a PKC degradation rate over a 24-h period (Physique 1, D and E) consistent with the normal turnover of the protein (Mashukova three-dimensional reconstructions of the confocal stacks. (B, D) The single apical (supranuclear) confocal sections approximately 1C1.5 CPDA m below the plasma membrane (resolution, 0.6 m). (E) Top section of the stack, showing images that include but are not restricted to the apical plasma membrane. Colocalizations were performed with other proteins in the green channel as follows: (A, B) keratin 8 (Krt8) and (C, D) FITC-transferrin by incubating the cells with the probe from the apical side overnight. (E) Rab11 (ARE marker). In the merged panels, colocalization images appear.