Daniel Caput

p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.

The p63 gene, a homologue of the tumour-suppressor p53 (refs 1–5), is highly expressed in the basal or progenitor layers of many epithelial tissues. Here we report that mice homozygous for a disrupted p63 gene have major defects in their limb, craniofacial and epithelial development. p63 is expressed in the ectodermal surfaces of the limb buds, branchial arches and epidermal appendages, which are all sites of reciprocal signalling that direct morphogenetic patterning of the underlying mesoderm. The limb truncations are due to a failure to maintain the apical ectodermal ridge, a stratified epithelium, essential for limb development. The embryonic epidermis of p63 −/− mice undergoes an unusual process of non-regenerative differentiation, culminating in a striking absence of all squamous epithelia and their derivatives, including mammary, lacrymal and salivary glands. Taken together, our results indicate that p63 is critical for maintaining the progenitor-cell populations that are necessary to sustain epithelial development and morphogenesis.

p63, a p53 Homolog at 3q27–29, Encodes Multiple Products with Transactivating, Death-Inducing, and Dominant-Negative Activities

We describe the cloning of p63, a gene at chromosome 3q27-29 that bears strong homology to the tumor suppressor p53 and to the related gene, p73. p63 was detected in a variety of human and mouse tissues, including proliferating basal cells of epithelial layers in the epidermis, cervix, urothelium, and prostate. Unlike p53, the p63 gene encodes multiple isotypes with remarkably divergent abilities to transactivate p53 reporter genes and induce apoptosis. Importantly, the predominant p63 isotypes in many epithelial tissues lack an acidic N terminus corresponding to the transactivation domain of p53. We demonstrate that these truncated p63 variants can act as dominant-negative agents toward transactivation by p53 and p63, and we suggest the possibility of physiological interactions among members of the p53 family.

Monoallelically Expressed Gene Related to p53 at 1p36, a Region Frequently Deleted in Neuroblastoma and Other Human Cancers

We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.

p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours.

p73 (ref. 1) has high homology with the tumour suppressor p53 (refs 2,3,4), as well as with p63, a gene implicated in the maintenance of epithelial stem cells. Despite the localization of the p73 gene to chromosome 1p36.3, a region of frequent aberration in a wide range of human cancers, and the ability of p73 to transactivate p53 target genes, it is unclear whether p73 functions as a tumour suppressor. Here we show that mice functionally deficient for all p73 isoforms exhibit profound defects, including hippocampal dysgenesis, hydrocephalus, chronic infections and inflammation, as well as abnormalities in pheromone sensory pathways. In contrast to p53-deficient mice, however, those lacking p73 show no increased susceptibility to spontaneous tumorigenesis. We report the mechanistic basis of the hippocampal dysgenesis and the loss of pheromone responses, and show that new, potentially dominant-negative, p73 variants are the predominant expression products of this gene in developing and adult tissues. Our data suggest that there is a marked divergence in the physiological functions of the p53 family members, and reveal unique roles for p73 in neurogenesis, sensory pathways and homeostatic control.

On the shoulders of giants: p63, p73 and the rise of p53.

The discoveries of the p53 homologs, p63 and p73, have both fueled new insights and exposed enigmas in our understanding of the iconic p53 tumor suppressor. Although the pivotal role of p53 in cancer pathways remains unchallenged, because p63 and p73 are now implicated in stem cell identity, neurogenesis, natural immunity and homeostatic control. Despite their seemingly separate tasks, there are hints that the p53 family members both collaborate and interfere with one another. The question remains, therefore, as to whether these genes evolved to function independently or whether their familial ties still bind them in pathways of cell proliferation, death and tumorigenesis.

Covalent Modification of p73α by SUMO-1 TWO-HYBRID SCREENING WITH p73 IDENTIFIES NOVEL SUMO-1-INTERACTING PROTEINS AND A SUMO-1 INTERACTION MOTIF

Two-hybrid screening in yeast with p73α isolated SUMO-1 (small ubiquitin-likemodifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif,hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73α, but not p73β, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73α is the C-terminal lysine (Lys627). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification siteb(X)XXhKXE, whereb is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73α on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.

Cloning and Characterization of a Specific Interleukin (IL)-13 Binding Protein Structurally Related to the IL-5 Receptor α Chain

Interleukin-13 (IL-13) is a cytokine secreted by activated T lymphocytes that shares many, but not all, biological activities with IL-4. These overlapping activities are probably due to the existence of common receptor components. Two proteins have been described as constituents of the IL-4 receptor, a ∼140-kDa glycoprotein (IL-4R) and the γ chain (γc) of the IL-2 receptor, but neither of these proteins binds IL-13. We have cloned a cDNA encoding an IL-13 binding protein (IL-13R) from the Caki-1 human renal carcinoma cell line. The cloned cDNA encodes a 380-amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The IL-13R shows homology with the IL-5 receptor, and to a lesser extent, with the prolactin receptor. COS-7 cells transfected with the IL-13R cDNA bind IL-13 with high affinity but do not bind IL-4. COS-7 cells co-transfected with the cloned IL-13R cDNA and IL-4R cDNA resulted in the reconstitution of a small number of receptors that recognized both IL-4 and IL-13. Reverse transcription-polymerase chain reaction analysis detected the receptor transcript only in cell lines known to bind IL-13.

Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors

Corticotrophin‐releasing factor (CRF) is the principal hypothalamic factor governing the pituitary‐adrenal axis, but the wide extra‐pituitary distribution of CRF and its receptors suggest a major role for this neuropeptide in the integration of the overall physiological and behavioral responses of an organism to stress. We have cloned a CRF receptor complementary DNA (cDNA) by expression in COS‐7 cells of a cDNA library from the AtT20 mouse pituitary tumour cell line. The cloned mouse cDNA was then used as a probe to isolate a human CRF receptor cDNA from a human brain cDNA library. The mouse and human cDNAs both encode 415 amino acid proteins that are 97% identical, containing seven putative transmembrane domains characteristic of G protein‐coupled receptors. The CRF receptor shows homology with the receptors for growth hormone‐releasing factor, vasoactive intestinal peptide, secretin, parathyroid hormone, and calcitonin. COS‐7 cells transfected with the mouse CRF receptor cDNA bind radiolabelled ovine CRF with high affinity and respond specifically to CRF by accumulation of intracellular cAMP. A 2.7 kb mRNA coding for the CRF receptor could be detected in AtT20 cells and human cortex tissue. PCR analysis also detected the receptor transcript in human pituitary, brainstem, and testis.

Molecular cloning of a levocabastine-sensitive neurotensin binding site

A search for sequences homologous to the neurotensin receptor cDNA in a rat hypothalamic library has identified a novel neurotensin receptor (NTR‐2). The 1539 bp cDNA encodes a 416 amino acid protein and shows highest homology to the previously cloned neurotensin receptor (NTR‐1) (64% homology and 43% identity). Binding and pharmacological studies demonstrate that NTR‐2 expressed in COS cells recognizes neurotensin (NT) with high affinity as well as several other agonists and antagonists. However, a fundamental difference was found; unlike NTR‐1, NTR‐2 recognizes, with high affinity, levocabastine, a histamine H1 receptor antagonist previously shown to compete with NT for low‐affinity binding sites in brain.

Cloning of the human IL-13Rα1 chain and reconstitution with the IL-4Rα of a functional IL-4/IL-13 receptor complex

The human homologue of the recently cloned murine IL‐13 binding protein (IL‐13Rα1) was cloned from a cDNA library derived from the carcinoma cell line CAKI‐1. The cloned cDNA encodes a 427 amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The human protein is 74% identical to the murine IL‐13Rα1, and 27% identical to the human IL‐13Rα2. CHO cells expressing recombinant hIL‐13Rα1 specifically bind IL‐13 (K d≈4 nM) but not IL‐4. Co‐expression of the cloned cDNA with that of IL‐4Rα resulted in a receptor complex that displayed high affinity for IL‐13 (K d≈30 pM), and that allowed cross‐competition of IL‐13 and IL‐4. Electrophoretic mobility shift assay showed that IL‐13 and IL‐4 were able to activate Stat6 in cells expressing both IL‐4Rα and IL‐13Rα1, while no activation was observed in cells expressing either one or the other alone.