We identified an essential Saccharomyces cerevisiae protein, Tap42, that associates with Sit4, a type 2A-related protein phosphatase, and with the type 2A phosphatase catalytic subunits. The association of Tap42 with the phosphatases does not require the previously identified phosphatase subunits. Genetic analysis suggests that Tap42 functions positively with both phosphatases. Mutations in TAP42 can confer almost complete rapamycin resistance. In addition, Tap42/Sit4 and Tap42/PP2A complex formation is regulated by nutrient growth signals and the rapamycin-sensitive Tor signaling pathway. These findings, combined with the defect in translation of the tap42-11 mutant at the nonpermissive temperature, suggest that Tap42, Sit4, and PP2A are components of the Tot signaling pathway.

Rapamycin inhibits the TOR kinases, which regulate cell proliferation and mRNA translation and are conserved from yeast to man. The TOR kinases also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis. We have analyzed gene expression in yeast cells exposed to rapamycin using arrays representing the whole yeast genome. TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator Gln3, and globally represses ribosomal protein expression. gln3 mutations were found to confer rapamycin resistance, whereas ure2 mutations confer rapamycin hypersensitivity, even in cells expressing dominant rapamycin-resistant TOR mutants. We find that Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation. In summary, our results reveal that the TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis, and amino acid permease stability.

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.

The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator. Recent studies have identified a p53 relative, termed p73. We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73␣ and -␤ isoforms to transactivate various p53-responsive promoters. We show that both p73␣ and p73␤ activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299. However, a number of significant differences were observed between p53 and p73 and even between p73␣ and p73␤. Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37°C. Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay. When we examined complex formation in transfected mammalian cells, we found that p73␣ coprecipitates with mutant but not wild-type p53. Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73. By cotransfecting p73␣ along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73␣. This decrease in transcriptional activity is correlated with the reduced ability of p73␣ to promote apoptosis in the presence of tumorderived p53 mutants. Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.A new gene family whose encoded products show significant sequence similarity to the tumor suppressor protein p53 have been identified (32,33,50,59,60,72). KET, the first to be identified, was cloned from a rat circumvallate taste papilla cDNA library (59). p73, the second identified from a COS cell cDNA library, encodes for at least two splicing variants, p73␣ and p73␤ (32, 33). Finally, the human homolog of KET, referred to as either p51 or p63, encodes at least six isoforms (p63␣/p51B/p73L, p63␤, p63␥/p51A, ⌬Np63␣, ⌬Np63␤, and ⌬Np63␥) that are expressed in a tissue-specific manner and harbor different transactivation potentials (50,60,72). It has been proposed that this family of proteins is ancestral to human p53, in that all show significant amino acid similarity in their C-terminal p53-unrelated extensions to the squid p53 protein (33,59,72).The p53 protein is modular and can be divided into at least four distinct domains: (i) the amino-terminal transcriptional transactivation domain (residues ϳ1 to 70) (6,7,15,55,68), (ii) the PXXP domain (residues ϳ61 to 94) (70), (iii) the sequence-specific DNA binding domain (residues ϳ102 to 292) (1, 27, 51, 71), and (iv) the carboxy-terminal regulatory and tetramerization domains (residues ϳ320 to 393 and ϳ320 to 360, respectively) (3,4,57,71). The various isoforms of p73 and p51/p63 display a modular structure similar to that o...

p63, a member of the p53 gene family, encodes multiple proteins that may either transactivate p53 responsive genes (TAp63) or act as a dominant-negative factor toward p53 and p73 (Delta Np63). p63 is expressed in many epithelial compartments and p63(-/-) mice fail to develop skin, prostate, and mammary glands among other defects. It has been previously shown that p63 is expressed in normal urothelium. This study reports that p63 is regulated in bladder carcinogenesis and that p63 expression is lost in most invasive cancers whereas papillary superficial tumors maintain p63 expression. Examination of bladder carcinoma cell lines reveals that certain lines derived from invasive carcinomas maintain expression of Delta Np63, as demonstrated by both immunoblotting and confirmed by isoform-specific quantitative reverse transcriptase-polymerase chain reaction. Another novel finding reported in this study is the fact that p63(-/-) mice develop a bladder mucosa epithelial layer yet fail to complete uroepithelial differentiation, producing a nontransitional default cuboidal epithelium. These data indicate that in contrast to the skin and prostate, p63 is not required for formation of a bladder epithelium but is indispensable for the specific differentiation of a transitional urothelium.