RESUMEN
p63, the major regulator of epithelial development/differentiation, is mutated in human ectodermal dysplasias, such as ankyloblepharon, ectodermal dysplasia and clefting (AEC). We recently identified that p63alpha physically associated with mRNA processing/splicing proteins. We previously showed that p63 mutations mapped to the sterile alpha-motif led to disruption of these interactions and modulated an aberrant splicing of keratinocyte growth factor receptor contributing into molecular mechanism underlying AEC phenotype. To further investigate the molecular mechanisms associated with AEC syndrome we established the cellular model for this disorder by stable introduction of mutated allele [L514F] of p63alpha into immortalized keratinocyte cells. We showed that mutated DeltaNp63alpha mediated an aberrant splicing of its own p63 mRNA transcript, which in turn led to accumulation of proteasome-resistant C-terminal truncated p63. The truncated p63 failed to associate with the C-terminal domain of RNA polymerase II through SRA4 protein and, therefore affected keratinocyte proliferation, differentiation and survival and may strongly contribute to AEC phenotype.
Asunto(s)
Empalme Alternativo/genética , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Mutación/genética , Receptores Notch/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Secuencia de Bases , Cisplatino/farmacología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Humanos , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Transducción de Señal/efectos de los fármacosRESUMEN
p63 mutations have been identified in several developmental abnormalities, including split-hand/foot malformation (SHFM). In this study, we demonstrate that the C-terminal domain of p63alpha associates with the E2 ubiquitin conjugating enzyme, Ubc9. A p63alpha mutation, Q634X, which naturally occurs in SHFM modulated the interaction of p63alpha with Ubc9 in yeast genetic assay. Furthermore, Ubc9 catalyzed the conjugation of p63alpha with small ubiquitin modifier-1 (SUMO-1), which covalently modified p63alpha in vitro and in vivo at two positions (K549E and K637E), each situated in a SUMO-1 modification consensus site (phiKXD/E). In addition, p63alpha mutations (K549E and K637E) abolished sumoylation of p63alpha, dramatically activated transactivation properties of TAp63alpha, and inhibited the dominant-negative effect of DeltaNp63alpha. These p63alpha mutations also affected the transcriptional regulation of gene targets involved in bone and tooth development (e.g., RUNX2 and MINT) and therefore might contribute to the molecular mechanisms underlying the SHFM phenotype.
Asunto(s)
Deformidades Congénitas del Pie/metabolismo , Deformidades Congénitas de la Mano/metabolismo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína SUMO-1/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Fibroblastos/citología , Regulación de la Expresión Génica , Genes Supresores de Tumor , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Fosfoproteínas/química , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Unión Proteica , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/química , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción , Transcripción Genética , Proteínas Supresoras de Tumor , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
p53 family members with a transactivation (TA) domain induce cell cycle arrest and promote apoptosis. However, DeltaNp63 isotypes lacking the TA-domain promote cell proliferation and tumorigenesis in vitro and in vgammavo. Although p53, TAp63 or TAp73 are stabilized upon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease and phosphorylation of DeltaNp63alpha in squamous cell carcinoma cells. Further work revealed that RACK1 physically associated with the p63alpha C-terminal domain through its WD40 domain. However, stratifin binds with phosphorylated DeltaNp63alpha in response to cisplatin. Upon DNA damage induced by cisplatin, stratifin mediated a nuclear export of DeltaNp63alpha into cytoplasm and then RACK1 targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase. Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and protein degradation of DeltaNp63alpha, respectively. Our data suggest that modification and down regulation of DeltaNp63alpha is one of the major determinants of the cellular response to DNA damage in human head and neck cancers.