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.2009 Jul;29(14):3953-63.
doi: 10.1128/MCB.00449-09. Epub 2009 May 18.

Stxbp4 regulates DeltaNp63 stability by suppression of RACK1-dependent degradation

Affiliations

Stxbp4 regulates DeltaNp63 stability by suppression of RACK1-dependent degradation

Yingchun Li et al. Mol Cell Biol.2009 Jul.

Abstract

p63, a member of the p53 tumor suppressor family, is essential for the development of epidermis as well as other stratified epithelia. Collective evidence indicates that DeltaNp63 proteins, the N-terminally deleted versions of p63, are essential for the proliferation and survival of stratified epithelial cells and squamous cell carcinoma cells. But in response to DNA damage, DeltaNp63 proteins are quickly downregulated in part through protein degradation. To elucidate the mechanisms by which DeltaNp63 proteins are maintained at relatively high levels in proliferating cells but destabilized in response to stress, we sought to identify p63 interactive proteins that regulate p63 stability. We found that Stxbp4 and RACK1, two scaffold proteins, play central roles in balancing DeltaNp63 protein levels. While Stxbp4 functions to stabilize DeltaNp63 proteins, RACK1 targets DeltaNp63 for degradation. Under normal growth conditions, Stxbp4 is indispensable for maintaining high basal levels of DeltaNp63 and preventing RACK1-mediated p63 degradation. Upon genotoxic stress, however, Stxbp4 itself is downregulated, correlating with DeltaNp63 destabilization mediated in part by RACK1. Taken together, we have delineated key mechanisms that regulate DeltaNp63 protein stability in vivo.

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Figures

FIG. 1.
FIG. 1.
Downregulation of ΔNp63 in keratinocytes causes cell cycle arrest and eventually leads to senescence. (A) Extracts of sf9 cells infected with baculoviruses expressing various p63 isoforms were subjected to SDS-PAGE and immunoblotting with p63 polyclonal antibodies that were raised against either TA or ΔN isoforms. The sizes of the molecular weight markers (in thousands) are indicated on the left. (B) Lysates of HaCaT cells transfected with control (Luc) or p63-specific (p63-1) siRNA were immunoblotted with pan-p63 (4A4), ΔNp63-specific, or p63α-specific antibodies as described. Actin serves as a loading control. Molecular weights (in thousands) are indicated on the left. (C) HaCaT cells were transfected with control (Luc) or p63-specific (p63-1 or p63-2) siRNAs. Cells were collected 3 days later and processed for FACS analysis. (D) HaCaT cells were stained for senescence-associated β-Gal activity 6 days after siRNA transfection.
FIG. 2.
FIG. 2.
The physical interaction between p63 and Stxbp4. (A) Exogenously expressed p63 and Stxbp4 interact with each other. H1299 cells were transfected with plasmids encoding Flag-tagged Stxbp4 and Myc-tagged ΔNp63α or ΔNp63β. Cell lysates were immunoprecipitated (IP) with anti-Myc antibody and immunoblotted with anti-Myc and anti-Flag antibodies, respectively. A total of 15% of the lysate was used in the input sample. (B) The WW domain of Stxbp4 and the PPPPY motif of ΔNp63 are required for Stxbp4 interaction with p63. Shown on top is a schematic illustration of the modular structure of Stxbp4 and ΔNp63β and corresponding deletion or point mutation constructs. H1299 cells were transfected with the indicated plasmids and processed for immunoblotting as shown in panel A. A total of 10% of the lysate was used in the input sample. (C) Endogenous p63 proteins interact with exogenous Stxbp4. A Flag-tagged Stxbp4 construct was transfected into Scaber and HaCaT cells and after 24 h, cell lysates were prepared and immunoprecipitated with anti-ΔNp63 antibody or control rabbit IgG, followed by immunoblotting with anti-Flag and anti-p63 (4A4) antibodies, respectively. A total of 2% of the lysate was used in the input sample. (D) Endogenous Stxbp4 and p63 interact. Lysates of HaCaT cells were immunoprecipitated with anti-ΔNp63, anti-p63α, or control rabbit IgG, followed by immunoblotting with anti-p63 (4A4) and anti-Stxbp4 antibodies, respectively. A total of 1% of the lysate was used as the input sample. (E) Stxbp4 can directly bind p63 in vitro as indicated by far-Western analysis. His-tagged p63 proteins were purified from baculovirus-infected sf9 cells, subjected to SDS-PAGE, and then visualized by silver staining (right panel). GST or GST-Stxbp4 proteins were expressed in DH5α cells by IPTG (isopropyl-β-d-thiogalactopyranoside) induction, then separated by SDS-PAGE, and stained by Coomassie blue (left panel). The same lysate was resolved on two parallel gels, which were transferred to a nitrocellulose membrane, denatured by 6 M guanidinium HCl, and renatured by serial dilutions of guanidinium HCl as described in Materials and Methods. The membrane was then incubated with or without 0.5 mg/ml purified His-tagged p63 (the middle two panels) and immunoblotted with anti-His antibody. Molecular weights (in thousands) are indicated on the left.
FIG. 3.
FIG. 3.
Stxbp4, like ΔNp63, is essential for keratinocyte proliferation. HaCaT human keratinocytes were transfected with control luciferase (Luc), two Stxbp4, or two p63 siRNAs, as indicated. Cells were collected 72 h later and processed for immunoblotting (A) or FACS analysis (B). The population of cells in the S phase is shown as an index of cell proliferation. Molecular weights (in thousands) are indicated on the left in panel A.
FIG. 4.
FIG. 4.
Stxbp4 regulates ΔNp63 protein stability. (A) HaCaT cells were transfected with siRNAs as described in the legend for Fig. 3. At 72 h, cells were collected and processed for immunoblotting (W.B.) to detect p63, Stxbp4, p53, and actin (top four panels). The RNA transcript levels of p63, stxbp4, and actin were analyzed by RT-PCR (bottom three panels). (B) The dose-dependent p63 destabilization by Stxbp4 siRNA is shown, using Stxbp4-2 siRNA as an example. (C) At 62 h after siRNA transfection, HaCaT cells were treated with 20 μg/ml cycloheximide (CHC) and collected at the indicated time points to detect p63, Stxbp4, and actin by Western blotting. The results after densitometric analysis and normalization based on actin levels were graphed (right). (D) At 42 h after siRNA transfection, HaCaT cells were treated with or without MG132 for 8 h and were subjected to immunoblotting. (E) Scaber cells were transfected with control luciferase (Luc) or two Stxbp4 siRNAs for 72 h and analyzed by immunoblotting. (F) U2OS cells were transfected with Flag-tagged ΔNp63α alone or together with an increasing amount of Flag-tagged Stxbp4. Cells were collected 44 h later and were processed for immunoblotting using the indicated antibodies. Molecular weights (in thousands) are indicated on the left of panels B, C, D, E, and F.
FIG. 5.
FIG. 5.
Itch-mediated ΔNp63 degradation is inhibited by Stxbp4, but endogenous Itch is unlikely to be involved in ΔNp63 degradation. (A) 293 cells were transfected with Flag-tagged ΔNp63α (0.1 μg) and Myc-tagged Itch at the indicated ratios (0.1 μg, 0.3 μg, 1 μg, and 1.5 μg). (B) 293 cells were transfected with Flag-tagged ΔNp63α (0.1 μg) and Myc-tagged Itch (0.3 μg), together with different amounts of Flag-tagged Stxbp4 plasmid (0.3 μg, 1 μg or 3 μg). At 48 h after transfection, cells shown in panels A and B were processed for immunoblotting to detect p63, Itch, Stxbp4, and actin, as indicated. (C) HaCaT cells were transfected with control (luciferase [Luc]), Stxbp4 (Stxbp4-1), or Itch (Itch-1 and Itch-2) siRNAs. Cells were collected 72 h later and processed for immunoblotting. Molecular weights (in thousands) are indicated on the left.
FIG. 6.
FIG. 6.
ΔNp63 destabilization in the absence of Stxbp4 is dependent on RACK1 pathway. (A) U2OS cells were transfected with 0.1 μg plasmid expressing Flag-tagged ΔNp63α alone and with increasing amounts of plasmid expressing T7-tagged RACK1 (0.3 μg, 1 μg, or 3 μg). Cells were collected 24 h later and processed for immunoblotting. (B) U2OS cells were transfected with Flag-tagged ΔNp63α (0.1 μg) and T7-tagged RACK1 (2 μg), together with increasing amounts of Flag-tagged Stxbp4 (0.5 μg, 1 μg, or 2 μg). At 40 h after transfection, cells were processed for immunoblotting using the indicated antibodies. (C) HaCaT cells were transfected with control (luciferase [Luc]), Stxbp4 (Stxbp4-1), RACK1 (Rack1-KD1) siRNAs, or siRNAs against both Stxbp4 and RACK1. Cells were processed for immunoblotting 72 h after transfection. Molecular weights (in thousands) are indicated on the left.
FIG. 7.
FIG. 7.
ΔNp63 and Stxbp4 are downregulated upon DNA damage. (A) HaCaT cells were treated with 300 nM camptothecin (CPT) for the indicated time points. Cells were collected for both immunoblotting (W.B.) (top two panels) and RT-PCR with primers specific for ΔNp63 or β-actin mRNAs (bottom two panels). (B) An H1299 Tet-off cell line was cultured in medium without tetracycline to induce ΔNp63α expression for 24 h. Cells were then treated with 300 nM CPT and 30 μΜ etoposide (ETP) for 21 h and processed for immunoblotting. (C and D) HaCaT cells were transfected with siRNAs for RACK1, Itch, or control (luciferase [Luc]). At 48 h after transfection, cells were treated with either 30 μΜ ETP or 300 nM CPT for 24 h and processed for immunoblotting using the indicated antibodies. DMSO, dimethyl sulfoxide. HaCaT cells were treated with either 30 μΜ ETP or 300 nM CPT for 24 h (E) or with 300 nM CPT for the indicated time points (F). Cells were processed for immunoblotting to detect Stxbp4, p63, and actin. Molecular weights (in thousands) are indicated on the left.
FIG. 8.
FIG. 8.
Models for the regulation of ΔNp63 stability under normal growth conditions and in response to DNA damage. (A) In resting conditions, ΔNp63 is expressed at a relatively high level to promote cell proliferation and/or survival. Its basal level is maintained by Stxbp4, which suppresses RACK1-mediated degradation. (B) Following DNA damage, Stxbp4 itself is downregulated, allowing RACK1 to target ΔNp63 for degradation, which eventually leads to cell cycle arrest or cell death.
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References

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