Loss of Nedd4-1 expression in MEFs derived from Nedd4-1 knockout mice. (A) Genotyping of MEFs generated from Nedd4-1^+/+, Nedd4-1^+/−trap, and Nedd4-1^−/−trap embryos. PCR analysis of genomic DNA from the indicated cells is shown. A 447-bp fragment corresponds to the WT allele and a 680-bp fragment to the trapped allele. (B) Immunoblotting of lysates from Nedd4-1^+/+, Nedd4-1^+/−trap, and Nedd4-1^−/−trap MEFs. Cell lysates were probed for Nedd4-1 and Nedd4–2 by using anti-Nedd4-1 antibodies (Upper) and anti-Nedd4–2 antibodies (Lower), respectively. Actin levels were determined with anti-actin antibodies. (C) Immunoblotting of lysates from Nedd4-1^+/+ and Nedd4-1^{−/−exons9,10} MEFs. Ten or 40 μg of proteins obtained from cell lysates of the indicated MEFs were probed for Nedd4-1 by immunoblotting with anti-Nedd4-1 antibodies, as in B. Actin levels were determined with anti-actin antibodies.

PTEN stability is not affected by Nedd4-1. (A) PTEN levels are unchanged in the absence (knockout) of Nedd4-1. Levels of PTEN in MEFs and heart tissue from Nedd4-1^+/+, Nedd4-1^+/−trap, and Nedd4-1^−/−trap embryos (Top), and MEFs from Nedd4-1^+/+ and Nedd4-1^{−/−exons9,10} embryos (Bottom) were analyzed by immunoblotting. Expression of Nedd4-1 in embryonic hearts of Nedd4-1^+/+ and Nedd4-1^+/−trap (but not Nedd4-1^−/−trap) mice is depicted in the Middle. (B and C) PTEN levels are unchanged by knockdown of Nedd4-1. (B) HEK293T cells were either not transfected or were transfected individually with three different shRNAmir (in pGIPZ) directed toward the human Nedd4-1 sequence or pGIPZ plasmid containing a nonsilencing control. After 72 h, cells were lysed, and levels of Nedd4-1, PTEN, and actin were determined by immunoblotting with their respective antibodies. Endogenous levels of Nedd4-1 were reduced 62%, 70%, and 71% for shRNA (), shRNA () and shRNA (), respectively (average of n = 5, relative to cells transfected with nonsilencing control). Average PTEN levels in these experiments remained close to 100% for each of the knockdown constructs [93%, 90%, and 97% for shRNA (), shRNA () and shRNA (), respectively (n = 5, relative to nonsilencing control)]. Actin levels remained constant as well (99%, 105%, and 102% compared with nonsilencing control, respectively). The experiment shown is a representative of three independent experiments, each performed in duplicate. (C) WT MEFs (Nedd4-1^{+/+,exons9,10}) were transfected with shRNA directed against mouse Nedd4-1. Cells were then analyzed for the extent of Nedd4-1 knockdown and levels of PTEN and actin, as described above. Average Nedd4-1 knockdown was 56% (n = 4), whereas actin levels remained unchanged. (D) Reconstitution of Nedd4-1 in Nedd4-1^{−/−exons9,10} MEFs does not alter PTEN levels. Nedd4-1^{−/−exons9,10} MEFs were transfected with GFP-Nedd4-1, and 24 h later cells were analyzed for expression of Nedd4-1 by using anti-GFP antibodies, and for the levels of PTEN and actin, as described above.

PTEN ubiquitination is comparable in Nedd4-1^+/+ and Nedd4-1^−/−trap MEFs and lack of association between PTEN and Nedd4-1. (A) PTEN ubiquitination: Nedd4-1^+/+ and Nedd4-1^−/−trap MEFs were pretreated with a proteasome inhibitor (20 μM MG132). They were then lysed, and the lysate was boiled in SDS to remove PTEN-associated proteins. After dilution of the SDS, PTEN was immunoprecipitated from the lysates, and immunoprecipitates were immunoblotted with anti-ubiquitin antibody to detect ubiquitinated PTEN (PTEN-Ub; Upper) or anti-PTEN antibody (Lower). Control (Ctr): Immunoprecipitate with beads alone (without anti-PTEN antibodies). The left two lanes represent total ubiquitination of the MEF lysates used for the experiment. (B) PTEN stability is unchanged in the absence of Nedd4-1. Nedd4-1^+/+ MEFs (Top Left) or Nedd4-1^−/−trap MEFs (Bottom Left) were exposed to cycloheximide (CHX) and the amount of PTEN remaining was analyzed over time (0, 3, 6, 9 h) by immunoblotting. Identical results were obtained in the Nedd4-1^{−/−exons9,10} MEFs (data not shown). Quantification of the pulse–chase data from both the Nedd4-1^−/−trap and Nedd4-1^{−/−exons9,10} MEFs (and their wild-type controls) is shown in the Right (mean± SD, n = 4). (C and D) PTEN does not bind Nedd4-1. (C) PTEN was immunoprecipitated from Nedd4-1^+/+ and Nedd4-1^−/−trap MEFs, and the precipitates were immunoblotted for Nedd4-1 (Upper) or PTEN (Lower) with their respective antibodies. Control (Ctr): beads alone (without anti-PTEN antibodies). (D) (Upper) Immobilized GST-PTEN, GST alone (negative control), or GST-LAPTM5(C terminus) (GST-LAPTM5-Cter; positive control) were incubated with lysates from Nedd4-1^+/+ and Nedd4-1^−/−trap MEFs, and the precipitate was immunoblotted for Nedd4-1. (Lower) GST fusion proteins (arrowheads) used for the pulldowns, analyzed by Ponceau S staining. Binding experiments were repeated two to four times with identical results.

PKB/Akt activation does not depend on Nedd4-1. Subconfluent Nedd4-1^+/+, Nedd4-1^+/−trap, and Nedd4-1^−/−trap MEFs (A) or Nedd4-1^+/+ and Nedd4-1^{−/−exons9,10} MEFs (B) were starved overnight followed by either no treatment, 10-min stimulation with 10% FBS, or 50 μM LY294002 treatment for 1 h followed by FBS stimulation. Equal amounts of protein lysates were immunoblotted with anti-phospho-PKB (Ser-473), anti-PKB, anti-PTEN, or anti-GAPDH antibodies. Results are representative of three independent experiments.

PTEN subcellular localization is independent of Nedd4-1. Nedd4-1^+/+ and Nedd4-1^−/−trap MEFs (A) or Nedd4-1^+/+ and Nedd4-1^{−/−exons9,10} MEFs (B) were immunostained for PTEN (red) and DAPI (blue, to mark the nucleus) and analyzed by confocal microscopy to evaluate cellular distribution of PTEN. (Scale bar: 16 μm.)

Nedd4-1 does not affect activation of the Rad51 promoter by PTEN. Nedd4-1^+/+, Nedd4-1^{−/−exons9,10}, or PC-3 cells were transiently transfected with the indicated plasmids, and lysates were assayed for luciferase activity. The results are represented as the mean ± SD (n = 4 samples, representative of three independent experiments). Luciferase activity is shown as fold induction normalized to basal Rad51 promoter activity. There is no statistically significant difference in Rad51 activation by PTEN between the WT and Nedd4-1(−/−) cells.