Regulation of p53 family member isoform DeltaNp63alpha by the nuclear factor-kappaB targeting kinase IkappaB kinase beta

Abstract

The p53 family gene p63 plays an instrumental role in cellular stress responses including responses to DNA damage. In addition to encoding a full-length transcriptional activator, p63 also encodes several dominant inhibitory isoforms including the isoform DeltaNp63alpha, the function of which is not fully understood. DeltaNp63alpha is degraded in response to DNA damage, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying regulation of DeltaNp63alpha expression in response to chemotherapeutic agents or tumor necrosis factor-alpha. We found that DeltaNp63alpha interacts with IkappaB kinase (IKK), a multisubunit protein kinase that consists of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. The IKKbeta kinase promotes ubiquitin-mediated proteasomal degradation of DeltaNp63alpha, whereas a kinase-deficient mutant IKKbeta-K44A fails to do so. Cytokine- or chemotherapy-induced stimulation of IKKbeta caused degradation of DeltaNp63alpha and augmented transactivation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, IKKbeta inhibition attenuated cytokine- or chemotherapy-induced degradation of DeltaNp63alpha. Our findings show that IKKbeta plays an essential role in regulating DeltaNp63alpha in response to extrinsic stimuli. IKK activation represents one mechanism by which levels of DeltaNp63alpha can be reduced, thereby rendering cells susceptible to cell death in the face of cellular stress or DNA damage.

Figure 1. IKK interacts with and regulates ΔNp63α in response to Cisplatin or TNF-α

(a) Isoform-specific real-time quantitative RT-PCR to detect isoforms TAp63α and ΔNp63α in JHU-022 cells (left panel). Western blot analysis of cellular lysates from either the p53/p63 deficient cell line H1299 or JHU-022 cells (right panel) that were untransfected or transfected with either TAp63α or ΔNp63α expression plasmids, as indicated. (b) Loss of ΔNp63α and nuclear accumulation of IKKβ and IKKα in JHU-022 cells in response to treatment with cisplatin. At the indicated time periods after treatment with cisplatin, JHU-022 cells were fractionated into nuclear and cytoplasmic fractions and analyzed by immunobloting with the indicated antibodies. (c) Complex formation between ΔNp63α with either IKKβ or IKKα. Panel i: JHU-022 cells were transfected with either HA-IKKα or Flag-IKKβ or empty Flag-vector. Whole cell lysates were immunoprecipitated with either anti-flag or anti-HA matrix, as indicated, and subjected to western blot analysis using either anti-p63 or anti-IKKα or anti-IKKβ antibodies, as indicated. Panel ii: J H U-022 cells were transfected with Flag-ΔNp63α or empty Flag–vector. Immunoprecipitation was performed using anti-flag matrix and the membrane was blotted with either anti-IKKβ, anti-IKKα, or anti-p63 antibody, as indicated. Panel iii: Immunoprecipitates of JHU-022 cell lysates using either anti-p63 or anti-IKKβ antibody were subjected to immunoblot analysis using either anti-IKKβ or anti-p63 antibody, as indicated. (d) IKK is required for TNF-α– or cisplatin-mediated reduction of ΔNp63α in JHU-022 cells. Panel i: JHU-022 cells were pre-treated with or without 100μM NEMO inhibitory peptide or vehicle control for 2h followed by treatment with or without 20 ng/ml TNF-α for 10h. Whole cell lysates were collected and subjected to Western blot using anti-p63 antibody. (lane 1 – untreated cells, lane 2 – TNF-α for 10h, lane 3 – TNF-α for 16h, lane 4 – NBD+TNF-α, lane 5 – NBD alone). Panel ii: JHU-022 cells were pre-treated with or without 100μM NEMO inhibitory peptide or vehicle control for 2h followed by treatment with or without cisplatin for 8h. Cells were harvested and subjected to Western blot using anti-p63 antibody.

Figure 2. IKKβ kinase mediates the reduction of ΔNp63α levels by cisplatin or TNF-α

(a,b and c) JHU-022 and p53/p63 deficient H1299 cells were transfected with increasing concentrations (0, 0.5, 1 and 1.5μg) of expression plasmids encoding either IKKβ (a), IKKβK44A (b), or IKKα (c) as indicated; Western blot was performed using the indicated antibodies. p53/p63 deficient H1299 cells were also transfected with 1μg of ΔNp63α expression plasmid. (d) p53/p63 deficient H1299 cells were transfected with 1μg of TAp63α expression plasmid with increasing concentrations (0, 0.5, 1 and 1.5μg) of expression plasmids encoding IKKβ and subjected to Western blotting with the indicated antibodies.

Figure 3. IKKβ is required for the reduction of endogeous ΔNp63α in response to cisplatin or TNF-α

(a) JHU-022 cells were transfected with increasing concentrations of either IKKβ RNAi plasmid or IKKα RNAi plasmid, and subjected to Western blot analysis using anti-p63 antibodies to assess the endogenous levels of p63. (b) JHU-022 cells were transfected with or without IKKβ RNAi plasmid or IKKα RNAi plasmid; 24h after transfection, the cells were treated with or without 75μM cisplatin for 8h, and cell lysates were subjected to Western blot using anti-p63 antibodies. (c) JHU-022 cells were transfected with or without IKKβ RNAi plasmid; 24h after transfection, the cells treated with or without TNF-α. Cells were harvested and lysates were subjected to Western blot using anti-63 antibody. (lane1 - untreated cells, lane 2 - TNF-α for 10h, lane 3 - TNF-α for 16h, lane 4 - IKKβ RNAi + TNF-α for 10h, lane 5 - IKKβ RNAi + TNF-α for 16h).

Figure 4. IKKβ promotes ubiquitin-mediated proteasomal degradation of ΔNp63α

(a) JHU-022 cells were transfected with or without an expression plasmid encoding IKKβ; 24h after transfection, cells were treated with 10μM MG132 for 10h. Whole cell lysates were subjected to Western Blot analysis using anti-IKKβ or anti-p63 antibodies. (b) JHU-022 cells were transfected with constant amount of ΔNp63α expression plasmid with or without expression plasmids encoding either IKKβ, IKKβ K44A, or empty vector, as indicated. 24h after transfection, the cells were treated with 100μg/ml cycloheximide. At the indicated time points, whole cell lysates were analyzed for ΔNp63α by immunoblotting. Actin was used for loading control. (c) JHU-022 cells were transfected with constant amount of IKKβ RNAi expression plasmid. 24h after transfection, the cells were treated with 100μg/ml cycloheximide. At the indicated time points, whole cell lysates were analyzed for endogenous ΔNp63α by immunoblotting. Actin was used for loading control. (d) JHU-022 cells were co-transfected with ΔNp63α and Ub-HA expression plasmids, with or without increasing concentrations of an expression vector encoding either IKKβ, IKKβK44A, IKKα or IKKαK44M. At 36h following transfection, cells were treated with MG132 for 10h. Cell lysates were immunoprecipitated with anti-HA-matrix and subjected to western blot analysis with an antibody that recognizes ΔNp63α to assess the ubiquitination levels of ΔNp63α.

Figure 5. IKKβ counteracts ΔNp63α-mediated repression of p53-dependent transactivation

(a) JHU-022 cells were transfected with either p21 or Bax luciferase promoter construct along with Renilla luciferase plasmid, with or without IKKβ and/or ΔNp63α or TAp63α, as indicated. The amount of DNA per transfection was kept constant by using empty pCDNA3.1 vector. At 24 h post-transfection, the luciferase activity was determined. The transfection efficiency was standardized against Renilla luciferase. Results shown are representative of three independent experiments. * indicated p ≤ 0.001. (b) JHU-022 cells were transfected with increasing concentrations of IKKβ expression plasmid and Western blot analysis was performed with the indicated antibodies to assess the endogenous levels of the indicated proteins. (c) JHU-022 cells were transfected with empty vector or expression vector encoding either TAp63α, ΔNp63α, or IKKβ (alone or in combination with each other, as indicated), and the endogenous levels of p21 and Bax protein levels were assessed by Western Blotting. (d) JHU-022 cells were transfected with either ΔNp63α, TAp63α, or IKKβ (alone or in combination with each other, as indicated) and analyzed after 24h for activation of caspase-9 (Western blot assay of caspase-9 cleavage product) and induction of apoptosis (measured using annexin V/PI staining).