Identification of DNp63a protein interactions by mass spectrometry

p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (Delta N) a transactivation domain. Delta Np63 alpha is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of Delta Np63 alpha, we set out to identify Delta Np63 alpha interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential Delta Np63 alpha binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of Delta Np63 alpha with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that Delta Np63 alpha exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of Delta Np63 alpha in mRNA metabolism and transport.