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

{Delta}Np63 is a master regulator of epithelial development, driving the expansion of progenitor cells in stratified epithelia. Mutations in {Delta}Np63 are linked to squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs), as well as to ectodermal dysplasia syndromes such as ectrodactyly-ectodermal dysplasia-clefting (EEC) and ankyloblepharon-ectodermal dysplasia-clefting (AEC). Although {Delta}Np63 functions as a nuclear transcription factor, the mechanisms underlying its nuclear import remain incompletely understood. By combining imaging, biochemical, structural and functional assays, we have thoroughly characterized {Delta}Np63 nuclear import, as mediated by the importin (IMP) /{beta}1 heterodimer. We also show here that {Delta}Np63 has evolved a peculiar strategy to ensure mutation tolerant nuclear localization, which is essential for DNA binding and transcriptional regulation. Despite a canonical bipartite NLS formed by two stretches of basic amino acids was identified between the DNA binding and oligomerization domains, each of them in sufficient to bind both IMP binding sites upon homodimerization. Therefore, in contrast to most known bipartite NLSs, only simultaneous substitution of both basic stretches of amino acids ablated nuclear localization, interaction with IMP, and decreased transcriptional activity. Since several {Delta}Np63 isoforms which lack the N-terminal basic stretch of amino acids have been described, and a number of mutations in the {Delta}Np63 NLS region have been identified in the Genome Aggregation Database, {Delta}Np63 has specifically evolved to tolerate mutations in its NLS without significantly compromising its ability to localize in the nucleus. GRAPHICAL ABSTRACT