The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation

Abstract

The tumor suppressor, PTEN is key to the regulation of diverse cellular processes, making it a prime candidate to be tightly regulated. The PTEN level is controlled in a major way by E3 ligase-mediated degradation through the Ubiquitin-Proteasome System (UPS). Nedd 4-1, XIAP, and WWP2 have been shown to maintain PTEN turnover. Here, we report that CHIP, the chaperone-associated E3 ligase, induces ubiquitination and regulates the proteasomal turnover of PTEN. It was apparent from our findings that PTEN transiently associates with the molecular chaperones and thereby gets diverted to the degradation pathway through its interaction with CHIP. The TPR domain of CHIP and parts of the N-terminal domain of PTEN are required for their interaction. Overexpression of CHIP leads to elevated ubiquitination and a shortened half-life of endogenous PTEN. On the other hand, depletion of endogenous CHIP stabilizes PTEN. CHIP is also shown to regulate PTEN-dependent transcription presumably through its down-regulation. PTEN shared an inverse correlation with CHIP in human prostate cancer patient samples, thereby triggering the prospects of a more complex mode of PTEN regulation in cancer.

FIGURE 1.

CHIP interacts and colocalizes with PTEN. HEK293 cells were seeded in 100-mm plates and transfected with or without 5 μg of His-PTEN. 100 μg of WCLs from control and His-PTEN were subjected to Ni-NTA pull-down assay. A, the Ni-NTA-purified sample was used for immunoblotting using CHIP, XIAP, WWP2, and Nedd4-1 antibodies. B, GST, GST-PTEN, and His-CHIP were separately expressed in BL21DE3 cells and used for a GST pull-down assay and IB with anti-CHIP and anti-GST antibodies. C and D, HEK293 cells were cotransfected with 2.5 μg each of GFP-PTEN and Flag-CHIP. Proteins were immunoprecipitated from WCLs with antibodies against GFP and Flag and checked for CHIP and PTEN, respectively. E, GFP-PTEN was overexpressed in HEK293 cells and pulled down with anti-PTEN antibody and probed for CHIP and PTEN. F, HeLa and DBTRG-05MG cells were stained with primary antibodies against PTEN and CHIP, followed by secondary antibodies conjugated to AF488 (PTEN, green) or AF594 (CHIP, red), and visualized under fluorescence microscope. Nuclei were visualized by DAPI staining. The images were acquired at 60× magnification. Cont: Control.

FIGURE 2.

Domains of PTEN and CHIP essential for their interaction. A, schematic representation of CHIP-WT and its generated mutants. B, HEK293 cells were cotransfected with: GFP-PTEN (2.5 μg) and 2.5 μg of either CHIP and its mutants TPR, ΔTPR, or ΔUbox (all Flag-tagged). WCLs were immunoprecipitated with anti-Flag antibody and IB with anti-PTEN and anti-Flag antibodies. C, 2.5 μg of CHIP and its deletion mutants, Δcc and Ubox were transfected along with GFP-PTEN (2.5 μg). WCLs were used for IP with FLAG antibody and IB with anti-PTEN and anti-FLAG antibodies. D, schematic diagram of PTEN-WT and its various truncated mutants. E, GFP-tagged PTEN-WT and its mutants, D1, D2, D3, and D4 as well as His-CHIP were separately expressed in HEK293 cells. His-CHIP (500 μg) was incubated with 500 μg of either of D1, D2, D3, or D4 for 2 h at 4 °C. The preincubated lysates were then subjected to IP with GFP antibody and probed for CHIP and GFP. HC: heavy chain.

FIGURE 3.

CHIP is an E3 ubiquitin ligase for PTEN. A–C, HEK293 cells were transfected with either HA-Ub or GFP-PTEN alone or in combination in the presence or absence of Flag-CHIP as shown in the figure, followed by treatment with or without MG132. IP was performed with either PTEN or Ub and IB against Ub, PTEN, or GFP, respectively. D, HEK293 cells were transfected with either scrambled siRNA (cont si), CHIP siRNA (CHIP si), Nedd4-1 siRNA (Nedd4-1 si), or WWP2 siRNA (WWP2 si). Whole cell lysates were pulled down with anti-PTEN antibody and probed for Ub. E, GFP-PTEN, Flag-CHIP, HA-ubiquitin, and Flag-H260Q were expressed separately in HEK293 cells. GFP-PTEN was then incubated with or without HA-Ub and in the presence or absence of either Flag-CHIP or Flag-H260Q and each of the incubated mixtures was then pulled down with PTEN and IB with Ub. F, GST-PTEN (rPTEN), GST-CHIP (rCHIP), and GST were purified using a GST column. The purified proteins were then checked by SDS-PAGE and stained with Coomassie Blue. GST or rPTEN were incubated for 4 h at 37 °C with or without rCHIP, F1, and F2 in the presence of Ub and ATP. The incubated mixtures were then immunoprecipitated with anti-Ub antibody and IB with PTEN.

FIGURE 4.

CHIP accounts for the proteasomal degradation of PTEN. HEK293 cells were transfected with: A, 2.5 μg of Flag-CHIP or Flag-H260Q, B, 2 μg of either PTEN mutants, C124A or G129E in the presence or absence of Flag-CHIP, IB was performed against anti-GFP. C, 30 nm of scrambled siRNA or CHIP siRNA. D and E, with or without similar amounts of Flag-CHIP or CHIP siRNA, followed by a treatment with 50 μm cycloheximide (CHX) for the time period indicated in the figure. The lysates were then checked for PTEN levels. F, EV or His-CHIP and treated with or without MG132. G, with 2 μg of GFP-PTEN, 1.5 μg of HA-Ub, 2 μg of Flag-CHIP, 1.5 μg of either HA-K48 or HA-K63 in combination as indicated in the figure. IP was performed with anti-PTEN antibody, and IB was carried out with anti-Ub antibody. All lysates were prepared post 48 h of transfection. EV: empty vector.

FIGURE 5.

Involvement of the chaperone system in the CHIP-mediated degradation of PTEN. HEK293 cells were used in these experiments. A, cells were treated with either the indicated concentrations of 17AAG or the vehicle (DMSO). The lysates were checked for Hsp90, Hsp70, PTEN. Cells were transfected with: B, GFP-PTEN, and the lysates were pulled down with either normal mouse IgG or PTEN antibody and probed for Hsp90, Hsp70, CHIP, and PTEN. C, EV or His-CHIP with or without treatment of 5 μm 17AAG and checked for PTEN status. D, cells were transfected with scrambled siRNA or CHIP siRNA and then treated with 5 μm 17AAG or equal amount of the vehicle, DMSO. Whole cell lysates were prepared and checked for PTEN level. E, 4 μg of GFP-PTEN, CHIP, K30A, and H260Q (all Flag-tagged) separately. Post-harvest, GFP-PTEN (500 μg) and 500 μg either of CHIP, K30A or H260Q were incubated together for 2 h at 4 °C, following which the incubation mix was immunoprecipitated with PTEN and IB against anti-Flag antibody. F, 2.5 μg of Flag-CHIP, Flag-K30A, or EV and was probed for PTEN. G, GFP-PTEN was co-expressed with either HA-Ub, Flag-CHIP, or Flag-K30A. The lysates were treated as mentioned under “Experimental Procedures” and then subjected to IP with anti-PTEN antibody and checked for Ub. In all immunoblotting experiments, actin served as the loading control. EV: empty vector.

FIGURE 6.

Functional activities of PTEN are regulated by CHIP. HEK293 cells were transfected with: A, either scrambled siRNA or CHIP siRNA, B, 2.5 μg of either Flag-CHIP or EV. The lysates were IB for pAKT (Ser-473). C, 4 μg of Flag-CHIP or EV, followed by subcellular fractionation. The levels of pAKT, AKT, PTEN, and CHIP were then probed. D, cDNA from HEK293, DU145, and PC3 cells were prepared and subjected to qPCR to check the endogenous transcript levels of PTEN and CHIP. E, PC3 cells were transfected with 2 μg of GFP-PTEN with either 2 μg of His-CHIP or the empty vector. cDNA was synthesized and qPCR performed to check the levels of Rad51, Dre1, Rbl2, VEGF, and c-Myc. F, HEK293 cells were transfected with PTEN and CHIP. Post-fixation, cells were stained with anti-BrdU-FITC antibody. PI was used as a DNA counterstain. Quadrant gates were applied to the PI (FL3-H) versus BrdU (FL1-H) dot plot, allowed for the discrimination of the cell subsets that were in proliferative/S phase (UL) of the cell cycle and had recently synthesized DNA, hence incorporated maximum BrdU. PI signal data were acquired in a linear mode as shown by the X-axis scale/FL3-H. G, immunohistochemistry was performed on human prostate hyperplasia (PH) and prostate cancer (PCa) samples to see the status of Ki67, CHIP, PTEN, and pAKT (Ser-473). Representative histological images of a section of the tumor stained with H&E was also provided. Pictures of all the sections were taken at 20× magnification except for Ki67 which was acquired at 40×. CE: cytoplasmic extract, NE: nuclear extract, ME: membrane extract, MtE: mitochondrial extract.