AEC-Associated p63 Mutations Lead to Alternative Splicing/Protein Stabilization of p63 and Modulation of Notch Signaling

Huang, Yi Ping; Kim, Yuriy; Li, Zhaobo; Fomenkov, Tanya; Fomenkov, Alexey; Ratovitski, Edward A.

doi:10.4161/cc.4.10.2086

Cited by 28 publications

(38 citation statements)

References 33 publications

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“…Mutations within the SAM domain responsible for AEC syndrome were originally described as inhibiting the transactivation of a p53 consensus promoter by TAp63, while also abolishing the ability of the Np63 isoform to inhibit p53-or TAp63-mediated transactivation (McGrath et al, 2001). The L514F (leucine 514 to phenylalanine) mutation within the SAM domain has been demonstrated to impair the ability of Np63 to bind and inhibit the splicing factors apobec-1-binding protein-1 (ABBP1) and SRA4, leading to aberrant spicing of components of the FGF and Notch signalling pathways and, indeed, of p63 itself (Candi et al, 2007b;Fomenkov et al, 2003;Huang et al, 2005). Mutations within the SAM domain of p63 have also been reported to prevent its direct binding and transcriptional regulation of the cell cycle checkpoint protein stratifin/14-3-3 (Fomenkov et al, 2003;Westfall et al, 2003).…”

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confidence: 99%

Differential altered stability and transcriptional activity of ΔNp63 mutants in distinct ectodermal dysplasias

Browne

Cipollone

Lena

et al. 2011

Journal of Cell Science

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SummaryHeterozygous mutations of p63, a key transcription factor in epithelial development, are causative in a variety of human ectodermal dysplasia disorders. Although the mutation spectrum of these disorders displays a striking genotype-phenotype association, the molecular basis for this association is only superficially known. Here, we characterize the transcriptional activity and protein stability of Np63 mutants (that is, mutants of a p63 isoform that lacks the N-terminal transactivation domain) that are found in ectrodactylyectodermal dysplasia-cleft syndrome (EEC), ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC) and nonsyndromic split-hand/split-foot malformation (SHFM). DNA-binding and sterile alpha motif (SAM) domain mutants accumulate in the skin of EEC and AEC syndrome patients, respectively, and show extended half lives in vitro. By contrast, C-terminal mutations found in SHFM patients have half-lives similar to that of the wild-type protein. The increased half-life of EEC and AEC mutant proteins was reverted by overexpression of wild-type Np63. Interestingly, the mutant proteins exhibit normal binding to and degradation by the E3 ubiquitin ligase Itch. Finally, EEC and AEC mutant proteins have reduced transcriptional activity on several skin-specific gene promoters, whereas SHFM mutant proteins are transcriptionally active. Our results, therefore, provide evidence for a regulatory feedback mechanism for p63 that links transcriptional activity to regulation of protein homeostasis by an unknown mechanism. Disruption of this regulatory mechanism might contribute to the pathology of p63-related developmental disorders.

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confidence: 99%

Differential altered stability and transcriptional activity of ΔNp63 mutants in distinct ectodermal dysplasias

Browne

Cipollone

Lena

et al. 2011

Journal of Cell Science

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“…Among these mRNA targets, we found RPN13 (also known ADRM1). We previously showed that cisplatin treatment induces the ATM-dependent phosphorylation of ⌬Np63␣, subsequently modulating various genes implicated in cell survival (33,(35)(36)(37). Using the isogenic HNSCC cell lines expressing wild-type ⌬Np63␣ or ⌬Np63␣-S385G (⌬Np63␣ with an altered ability to be phosphorylated by ATM kinase), we previously showed the failure of ⌬Np63␣-S385G to regulate gene transcription (36,37).…”

Section: Resultsmentioning

confidence: 99%

“…Cells and Reagents-We used HNSCC stable cell lines expressing wild-type ⌬Np63␣ or ⌬Np63␣-S385G (with an altered ability to be phosphorylated by ATM kinase) as described previously (33,(35)(36)(37). Cells were maintained in RPMI 1640 medium and 10% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were incubated with 10 g/ml cis-diamminedichloroplatinum (cisplatin, Sigma), 25 M lactacystin ␤-lactone (Calbiochem), or 20 units/ml human IFN␥ (Roche Diagnostics) for the indicated periods of time as described previously (22,55). Cells were lysed in 50 mM Tris (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 0.5% Brij-50, 1 mM PMSF, 0.5 mM NaF, 0.1 mM Na 3 VO 4 , and 2ϫ protease inhibitor mixture; sonicated five times for 10-s intervals; and clarified for 30 min at 15,000 ϫ g. Supernatants (total lysates) were used for immunoprecipitation and immunoblotting (33,(35)(36)(37). Both control (scrambled) siRNA and RPN13 siRNA (sc-72453) were obtained from Santa Cruz Biotechnology.…”

Section: Methodsmentioning

confidence: 99%

“…Because of several alternative splicing events, TP63 produces three isoforms with various lengths of the C terminus (␣, ␤, and ␥). ⌬Np63␣ is the longest TP63 protein among the ⌬Np63 isoforms and is the most predominant isoform expressed in HNSCC cells (35)(36)(37). We previously showed the importance of the ATM (ataxia telangiectasia mutated)-dependent phosphorylation of TP63 for its transcriptional activity in HNSCC cells upon cisplatin exposure (36,37).…”

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Phosphorylated TP63 Induces Transcription of RPN13, Leading to NOS2 Protein Degradation

Huang

Ratovitski

2010

Journal of Biological Chemistry

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Head and neck squamous cell carcinoma cells exposed to cisplatin display ATM-dependent phosphorylation of the most predominant TP63 isoform (⌬Np63␣), leading to its activation as a transcription factor. Here, we found that the phospho⌬Np63␣ protein binds to the genomic promoter of RPN13 through the TP63-responsive element. We further found that the phospho-⌬Np63␣ protein associates with other transcription factors (DDIT3 (also known as CHOP), NF-Y, and NF-B), activating RPN13 gene transcription. Furthermore, cisplatin-induced and phospho-⌬Np63␣-dependent RPN13 gene transcription leads to NOS2 degradation. Finally, we show that RPN13 knockdown by siRNA essentially rescues NOS2 from cisplatin-dependent inactivation. These data provide a novel mechanism for the phospho-⌬Np63␣-dependent regulation of NOS2 function in cells upon cisplatin treatment, contributing to the cell death pathway of tumor cells.Misfolding and aggregation of proteins may play an important part in the pathogenesis of cancer because cells utilize a physiologic aggresome pathway targeting various proteins into proteasome-dependent degradation in response to stress (1-4). There is emerging evidence that inhibiting the aggresome pathway leads to accumulation of misfolded proteins and to cell death (apoptosis or autophagy) in tumor cells (1-4). One of these misfolded proteins, inducible NOS2, was shown to be targeted into an aggresome/proteasome pathway, which leads to termination of NO production by NOS2 (5-8). NOS2 plays a critical role in massive NO production in a variety of cell types under physiologic and pathophysiologic conditions (9 -13). Understanding the molecular and cellular processes responsible for controlling NO production by NOS2 is critical for devising therapeutic strategies for many pathologic conditions, including cancer (14 -20).We previously found that the NAP110 (NOS2-associated protein of 110 kDa; also known as ADRM1 (adhesion regulatory molecule 1) or RPN13 (regulatory particle non-ATPase subunit 13)) forms protein-protein complexes with NOS2. This interaction modulates NOS2 activity by blocking its homodimerization (21, 22) and likely targeting NOS2 into a membrane fraction of cells, as reported by others (23). RPN13 is predominantly expressed as a non-glycosylated 42-kDa protein and is the ortholog of the yeast proteasome subunit . Furthermore, RPN13 functions as a novel 19 S proteasome cap-associated protein, acting as a receptor for ubiquitin, and recruits the deubiquitinating enzyme UCH37 to the 26 S proteasome (24 -27).Although RPN13 gene expression can be potentially induced by IFN␥ in gastric cancer cells, its transcriptional regulatory machinery is largely unknown (28). We thus undertook the study of transcription factors (TFs) 2 implicated in RPN13 gene regulation in head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin, the most used agent in chemotherapy for human cancers (29 -32). Among a few of the TFs controlling RPN13 gene transcription, we found TP63 (tumor protein 63).The TP53 homolog TP...

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Differential PERP regulation by TP63 mutants provides insight into AEC pathogenesis

Beaudry

Pathak

Koster

et al. 2009

American J of Med Genetics Pt A

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Ankyloblepharon Ectodermal Dysplasia and Cleft Lip/Palate (AEC) or Hay-Wells Syndrome is an autosomal dominant disorder characterized by a variety of phenotypes in ectodermal derivatives, including severe skin erosions, ankyloblepharon, coarse and wiry hair, scalp dermatitis, and dystrophic nails. AEC is caused by mutations in the gene encoding the TP63 transcription factor, specifically in the Sterile Alpha Motif (SAM) domain. The exact mechanism, however, by which these specific TP63 mutations lead to the observed spectrum of phenotypes is unclear. Analysis of individual TP63 target genes provides a means to understand specific aspects of the phenotypes associated with AEC. PERP is a TP63 target critical for cell-cell adhesion due to its participation in desmosomal adhesion complexes. As PERP null mice display symptoms characteristic of ectodermal dysplasia syndromes, we hypothesized that PERP dysfunction might contribute to AEC. Using luciferase reporter assays, we demonstrate here that PERP induction is in fact compromised with some, but not all, AEC-patient derived TP63 mutants. Through analysis of skin biopsies from AEC patients, we show further that a subset of these display aberrant PERP expression, suggesting the possibility that PERP dysregulation is involved in the pathogenesis of this disease. These findings demonstrate that distinct AEC TP63 mutants can differentially compromise expression of downstream targets, providing a rationale for the variable spectra of symptoms seen in AEC patients. Elucidating how specific TP63 target genes contribute to the pathogenesis of AEC will ultimately help design novel approaches to diagnose and treat AEC.

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AEC-Associated p63 Mutations Lead to Alternative Splicing/Protein Stabilization of p63 and Modulation of Notch Signaling

Cited by 28 publications

References 33 publications

Differential altered stability and transcriptional activity of ΔNp63 mutants in distinct ectodermal dysplasias

Differential altered stability and transcriptional activity of ΔNp63 mutants in distinct ectodermal dysplasias

Phosphorylated TP63 Induces Transcription of RPN13, Leading to NOS2 Protein Degradation

Differential PERP regulation by TP63 mutants provides insight into AEC pathogenesis

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