SUMMARY The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knock-in mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p5325,26, is severely compromised for transactivation of most p53 target genes and cannot induce G1-arrest or apoptosis in response to acute DNA damage. Surprisingly, p5325,26 retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p5325,26,53,54 mutant cannot induce senescence or inhibit tumorigenesis, like p53-nullizygosity. Thus, p53 transactivation is essential for tumor suppression, but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression -- a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression.

Sprouty negatively modulates branching morphogenesis in the Drosophila tracheal system. To address the role of mammalian Sprouty homologues in angiogenesis, another form of branching morphogenesis, a recombinant adenovirus engineered to express murine Sprouty-4 selectively in endothelial cells, was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Sprouty-4 expression inhibited branching and sprouting of small vessels, resulting in abnormal embryonic development. In vitro, Sprouty-4 inhibited fibroblast growth factor and vascular endothelial cell growth factor-mediated cell proliferation and migration and prevented basic fibroblast growth factor and vascular endothelial cell growth factor-induced MAPK phosphorylation in endothelial cells, indicating inhibition of tyrosine kinase-mediated signaling pathways. The ability of constitutively activated mutant Ras L61 to rescue Sprouty-4 inhibition of MAPK phosphorylation suggests that Sprouty inhibits receptor tyrosine kinase signaling upstream of Ras. Thus, Sprouty may regulate angiogenesis in normal and disease processes by modulating signaling by endothelial tyrosine kinases.The Drosophila tracheal (respiratory) system and the mammalian lung are both formed by patterned branching morphogenesis (1). In Drosophila, Branchless, a homologue of mammalian FGFs, 1 is required for normal tracheal branch patterning (2). Branchless activates Breathless, an FGF receptor homologue, inducing tracheal cell migration and branching (1-4). Loss of function mutations in the Drosophila sprouty gene, which encodes a 63-kDa protein containing a cysteinerich domain that is highly conserved in three human and four mouse homologues (5-8), cause enhanced tracheal branching (5). Further, inhibition of murine Sprouty-2 by antisense oligonucleotides enhances terminal branching of mouse cultured lung (6). These data suggest that Sprouty proteins negatively modulate branching morphogenesis in the Drosophila and mouse respiratory systems.Drosophila sprouty is expressed at the tips of growing primary branches of the tracheal system and also in other tissues such as eye imaginal disc, embryonic chordotonal organ precursors, and midline glia (5, 9, 10). Mouse homologues of sprouty (mSpry1, mSpry2, and mSpry4) are expressed in embryonic and adult tissues such as brain, heart, kidney, lung, limbs, and skeletal muscle (6 -8). FGF induces expression of sprouty in Drosophila and the chick embryo (5, 8), and Drosophila Sprouty is a membrane-associated protein that functions as a feedback inhibitor of the FGF signaling pathway (5, 9). Drosophila Sprouty can also antagonize epidermal growth factor signaling pathways (9 -11), and overexpression of Sprouty can cause phenotypes resembling those of loss, or reduction, of function of FGF and epidermal growth factor signaling pathways (5, 9 -11). Because angiogenesis shows some morphological similarities to branching of the Drosophila tracheal system and because it too requires receptor tyrosine kinase signals, including those mediated...

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Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play a key role in growth factor signaling pathways, although its signaling mechanism has not been fully elucidated. Using the yeast interaction trap system, we have identified Grb2 as a PI 3-kinase interacting protein. Our experiments demonstrate that p85, the regulatory subunit of PI 3-kinase, interacts with Grb2 in vivo, and this interaction is independent of growth factor stimulation. The direct association between Grb2 and p85 was reconstituted in vitro with glutathione S-transferase fusion proteins. Domain analyses and peptide competition indicate that the association is mediated by the SH3 domains of Grb2 and the proline-rich motifs of p85 and that only one SH3 domain is required for minimal binding. The interaction does not displace the catalytic subunit of PI 3-kinase but is exclusive of Sos. Signaling through PI 3-kinase, therefore, may involve the ubiquitous adapter Grb2, which serves as a convergence point for multiple pathways.