A bipartite, mutation-tolerant NLS regulates interaction of ΔNp63α with importin alpha, nuclear transport and transcriptional activity

Abstract ΔNp63α is a master regulator of epithelial development, driving the expansion of progenitor cells in stratified epithelia. Mutations in ΔNp63α are associated with squamous cell carcinomas (SCCs), basal cell carcinomas (BCCs), and ectodermal dysplasia syndromes, including ectrodactyly-ectodermal dysplasia-clefting (EEC) and ankyloblepharon-ectodermal dysplasia-clefting (AEC). Although ΔNp63α functions as a nuclear transcription factor, the mechanisms governing its nuclear import remain poorly understood. Here, by combining imaging, biochemical, structural, and functional approaches, we demonstrate that ΔNp63α nuclear import is mediated by the importin (IMP) α/β1 heterodimer. We also show that ΔNp63α has evolved a robust and mutation-tolerant mechanism to preserve nuclear localization, which is required for DNA binding and transcriptional activity. Despite the identification of a canonical bipartite nuclear localization signal (NLS), comprising two stretches of basic amino acids located between the DNA binding and oligomerization domains, each stretch is independently sufficient to engage both IMPα binding sites upon homodimerization. Consequently, unlike most known bipartite NLSs, only simultaneous substitution of both stretches abolished IMPα binding, impaired nuclear accumulation, and thereby prevented DNA binding and transcriptional activity. Given that several p63 isoforms lacking the N-terminal basic stretch have been described, and multiple variants within the NLS region are present in the Genome Aggregation Database, p63 appears to have evolved a resilient nuclear import mechanism that preserves nuclear localization despite mutations affecting its NLS.