Identification of ΔNp63r Protein Interactions by Mass Spectrometry Angela Amoresano, † Antonella Di Costanzo, ‡ Gabriella Leo, † Ferinando Di Cunto, § Girolama La Mantia, ‡ Luisa Guerrini, | and Viola Calabro `* ,‡ Dipartimento di Chimica Organica e Biologica, Universita ` Federico II, Napoli, Italy, Dipartimento di Biologia Strutturale e Funzionale, Universita ` Federico II, Napoli, Italy, Centro di Biotecnologie Molecolari, Universita ` di Torino, Torino, Italy, and Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita ` di Milano, Italy Received December 4, 2009 Abstract: p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal dif- ferentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (ΔN) a transactivation domain. ΔNp63R 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 ΔNp63R, we set out to identify ΔNp63R interacting pro- teins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential ΔNp63R binding proteins, including several heteroge- neous ribonucleoproteins (hnRNPs), were identified. In- tegration of the proteomic data with a Human Coexpres- sion 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 ΔNp63R with hnRN- PQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that ΔNp63R exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of ΔNp63R in mRNA metabolism and transport. Keywords: Epithelial Differentiation • Mass Spectrometry • p53 Gene Family • mRNA Metabolism • Protein Interaction Introduction The tumor suppressor p53 controls a powerful stress re- sponse by integrating upstream signals from different types of DNA damage and oncogenic stimuli. Activated p53 elicits cell cycle arrest and apoptosis, thereby preventing the formation of tumors. 1 p53 is the founding member of a family of proteins including p63 and p73. 2 All three genes can regulate cell cycle and apoptosis after DNA damage. However, mouse knockout models revealed an unexpected functional diversity among them. Indeed, p63 and p73 null mice exhibit severe develop- mental abnormalities but no increased cancer susceptibility, whereas this picture is reversed for p53 knockouts. 3 The structure of the corresponding genes, named TP53, TP63 and TP73, is evolutionarily conserved. In particular, the most conserved regions are those encoding the aminoterminal transactivation domain (TA), the central DNA binding domain (DBD), and the carboxyterminal oligomerization domain (OD). 2,3 The TP63 gene has two distinct promoters allowing the synthesis of proteins that either contain (TA) or lack (ΔN) a transactivation domain. In addition, alternative splicing at the 3′ end generates proteins with different C-termini, denoted R,  and γ. Only the R-isoforms (TA and ΔN) contain a Sterile Alpha Motif (SAM) domain, 4 which is absent in p53. Distinct p63 isoforms are expressed and differentially modulated during epithelial differentiation. 5 In particular, ΔNp63R is strongly expressed in basal keratinocytes of stratified epithelia and disappears in differentiating cells. Accordingly, human ΔNp63R is known to support the regenerative capacity of basal kerati- nocytes. 4 Heterozygous germ line mutations of p63 cause rare autosomal dominant developmental disorders associated with ectodermal dysplasia and limb deformities. 3 Accordingly, mice lacking p63 are severely compromised in their ability to generate limbs, and stratified epithelia such as skin. 2 In stark contrast with the high mutation rate of p53 in a large compendium of cancer types, p63 is not mutated in tumors. Therefore, although p63 is able to activate p53 responsive genes and induce apoptosis, it is debated whether it might act as a tumor suppressor or an oncogene. Remarkably, ΔNp63R gene is upregulated or amplified in a broad range of squamous cell carcinomas, thus, suggesting that p63 might be required to provide cancer cell population with a selective advantage. 6 Studies from different groups showed that ΔNp63R overex- pression contributes to cell dedifferentiation and induction of metastasis. 7,8 Conversely, it has recently been shown that complete loss of p63 is associated with cancer metastasis and poor prognosis, thus, implying that p63 can be a potential marker for cancer progression and invasion. 9 Despite its pleiotropic involvement in multiple facets of epithelial cell biology, mechanistic explanation of how p63 accomplishes its functions remains to be defined. The study of protein-protein interaction by mass spectrometry is an increasingly important strategy to understand the role of * To whom correspondence should be addressed. Prof. Viola Calabro `, Dipartimento di Biologia Strutturale e Funzionale, Universita ` Federico II, Napoli. Via Cinzia, Monte S Angelo, 80126 Napoli, Italy. Phone: +39 081 679069. Fax: +39 081 679033. E-mail: vcalabro@unina.it. † Dipartimento di Chimica Organica e Biologica, Universita ` Federico II. ‡ Dipartimento di Biologia Strutturale e Funzionale, Universita ` Federico II. § Universita ` di Torino. | Universita ` di Milano. 10.1021/pr9011156  XXXX American Chemical Society Journal of Proteome Research XXXX, xxx, 000 A PAGE EST: 6.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 ohio2/ypr-ypr/ypr-ypr/ypr99907/ypr3822d07z xppws 23:ver.5 1/30/10 0:36 Msc: pr-2009-011156 TEID: dmr00 BATID: 00000