Figure 3.

p34 promotes switching between NEDD4-1-mediated PTEN mono- and poly-ubiquitination. (a) Intact-cell ubiquitination assays revealed that PTEN is mono-ubiquitinated in p34-knockdown cells (MCF7, MDA-MB-231, DU145, and LS1034). (b) PTEN is localized to the nuclei of p34-knockdown cells. MCF7 cells were transfected with p34-siRNA for 24 h, fractionated into nuclear and cytosolic compartments, and analyzed for PTEN expression using western blot analyses. (c) p34 expression levels dictate the switch from PTEN poly-ubiquitination to mono-ubiquitination. PTEN ubiquitination in intact cells is significantly decreased by p34 and NEDD4-1 knockdown. The cells were transfected with p34-siRNA, NEDD4-1-siRNA, and/or p34 for 24 h. PTEN ubiquitination was determined using western blot analyses of immunoprecipitated PTEN-Ub adducts. (d) Cell-free ubiquitination assays of GST-PTEN that had been incubated with NEDD4-1 in the presence or absence of p34 revealed that PTEN is mono-ubiquitinated in the presence of NEDD4-1 alone and poly-ubiquitinated in the presence of both NEDD4-1 and p34. (e) PTEN poly-ubiquitination in cell-free assays decreases with decreasing doses of p34