The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the antiapoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component K D 535 ؎ 24 nM. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.A major function of the p53 tumor suppressor is the induction of an apoptotic program in response to a broad variety of cell stresses. Thus, understanding the mechanisms by which p53 executes cell death pathways is of considerable importance in cancer biology. The basis for the powerful apoptotic and tumor suppressor activity of p53 lies in its pleiotropism, which includes transcription-dependent and transcription-independent functions (1, 2). p53-mediated apoptosis primarily signals through the mitochondrial pathway (1). Some notable p53 target genes such as the BH3-only proteins PUMA, Noxa, Bax, and p53AIP1 reside and/or act at the mitochondria (3-7).We previously showed that in response to a death stimulus such as DNA damage or hypoxia, a fraction of stabilized p53 rapidly translocates to mitochondria in primary, immortal, and transformed cells (8 -11). The functional consequences of this phenomenon were revealed by targeting exogenous p53 to mitochondria in p53 null cells. Mitochondrially targeted p53 was sufficient to launch apoptosis and suppress colony formation directly from the mitochondrial platform in a transcription-independent fashion (9, 10). Translocated endogenous mitochondrial p53 interacts with anti-apoptotic BclXL and Bcl2 proteins and blocks their functions. Purified p53 protein induces oligomerization of Bak and permeabilization of the outer mitochondrial membrane and strongly promotes cytochrome c release from healthy unstressed mitochondria (10).Using computational and mutational analyses, we ...

Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

Macrophage migration-inhibitory factor (MIF) is an upstream regulator of innate immunity and a potential molecular link between inflammation and cancer. The unusual structural homology between MIF and certain tautomerases, which includes both a conserved substrate-binding pocket and a catalytic N-terminal proline (Pro1), has fueled speculation that an enzymatic reaction underlies MIF's biologic function. To address the functional role of the MIF tautomerase activity in vivo, we created a knock-in mouse in which the endogenous mif gene was replaced by one encoding a tautomerase-null, Pro13Gly1 MIF protein (P1G-MIF). While P1G-MIF is completely inactive catalytically, it maintains significant, albeit reduced, binding to its cell surface receptor (CD74) and to the intracellular binding protein JAB1/CSN5. P1G-MIF knock-in mice (mif P1G/P1G ) and cells derived from these mice show a phenotype in assays of growth control and tumor induction that is intermediate between those of the wild type (mif ؉/؉ ) and complete MIF deficiency (mif ؊/؊ ). These data provide genetic evidence that MIF's intrinsic tautomerase activity is dispensable for this cytokine's growth-regulatory properties and support a role for the N-terminal region in protein-protein interactions.Macrophage migration-inhibitory factor (MIF) is a widely expressed cytokine and upstream regulator of the immune response (23). Immunoneutralization and genetic knockout studies have established a central position for MIF in the host response to infection and tissue invasion (5, 9, 15). MIF's importance in human disease also has been revealed by the association of high-expression MIF alleles with clinical severity of different autoimmune disorders (18).An important role for MIF in tumorigenesis and in the contribution of inflammation to cancer development also has been proposed (7,20). Different tumor types express high levels of MIF, and clinical studies have shown that MIF production correlates with tumor aggressiveness and metastatic potential (1,22,27). Studies using genetically engineered MIFdeficient cells and mice show that MIF contributes to the development of the malignant phenotype by several mechanisms, including enhancement of cell cycle progression by sustained mitogen-activated protein kinase (MAPK) activation (28, 30), decreased proteasomal protein degradation (33) leading to altered expression of key cell cycle-regulatory proteins (15,21,35), and tumor promotion by neoangiogenesis (10, 48). Importantly, MIF also inhibits the proapoptotic and cell cycleregulatory function of the p53 tumor suppressor, thereby allowing for the accumulation of oncogenic mutations (20, 32). MIF's role in tumor progression additionally is supported by human genetic studies, and a recent report has described an association between high-expression MIF alleles and incidence of prostate cancer, which is a tumor type in which recurrent inflammation is considered to have an etiologic role (27).Information regarding MIF structure and function has emerged only in the last few year...

SUMMARY The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of line-age-specific genes, independent of p53.