Phosphorylation at Ser-15 may be a critical event in the up-regulation and functional activation of p53 during cellular stress. In this report we provide evidence that the ATM-Rad3-related protein ATR regulates phosphorylation of Ser-15 in DNA-damaged cells. Overexpression of catalytically inactive ATR (ATR ki ) in human fibroblasts inhibited Ser-15 phosphorylation in response to ␥-irradiation and UV light. In ␥-irradiated cells, ATR ki expression selectively interfered with late-phase Ser-15 phosphorylation, whereas ATR ki blocked UV-induced Ser-15 phosphorylation in a time-independent manner. ATR phosphorylated p53 at Ser-15 and Ser-37 in vitro, suggesting that p53 is a target for phosphorylation by ATR in DNA-damaged cells.

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.

The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3‐kinases. This study demonstrates that mTOR is a component of a cytokine‐triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor‐4E (eIF‐4E) binding protein, PHAS‐1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF‐4E‐dependent translation initiation. A mutant YAC‐1 T lymphoma cell line, which was selected for resistance to the growth‐inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS‐1 phosphorylation. In contrast, the PI 3‐kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS‐1 in both rapamycin‐sensitive and ‐resistant T cells. At similar drug concentrations (0.1–1 microM), wortmannin irreversibly inhibited the serine‐specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3‐kinase inhibitor, LY294002, at concentrations (1–30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85‐p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3‐kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.

The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.

These findings suggest that control of complement may underlie accommodation, at least in part, and raise the possibility that this control and possibly other protective mechanisms could be exerted by heparan sulfate.

Several independent studies indicate that synthetic inhibitors of cyclic-3',5'-nucleotide phosphodiesterase (PDE) isozymes, especially inhibitors of PDE-IV, are potent agents which suppress generation of reactive oxygen metabolites (ROM) by NADPH oxidase in leukocytes. Recent studies also show that NADPH oxidase is present in all cell types populating glomeruli. In view of this, we investigated PDE isozymes and their relation to ROM in isolated rat glomeruli. Glomeruli have the capacity to hydrolyze cAMP by isozymes PDE-II, PDE-III and PDE-IV, whereas cGMP is hydrolyzed by PDE-I and PDE-V. Inhibitor of PDE-IV rolipram inhibited significantly (cca 40 to 50%) ROM generation in response to stimulation by phorbol myristate acetate (PMA). Inhibitor of PDE-III cilostamide had only minor suppressive effects and inhibitors of other PDE isozymes did not influence ROM generation. Rolipram (3 microM) suppressed ROM generation without detectable increase in cAMP content. Incubation of glomeruli with forskolin, which increased cAMP content in glomeruli tenfold, inhibited ROM generation to a similar degree as rolipram. The suppression of ROM generation by rolipram was prevented by Rp-cAMPS, a specific inhibitor of protein kinase A (PKA) activity. Further, incubation of glomeruli with rolipram elicited marked in situ activation of PKA (+ 100%), as documented by increase in the (-cAMP/+cAMP) PKA activity ratio. We suggest that selective inhibitor of PDE-IV rolipram acted via the cAMP-signaling pathway and suppressed ROM generation possibly via phosphorylating ras-type GTP-binding protein component of NADPH oxidase and thereby blocking assembly of functional NADPH oxidase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract-Acute vascular or humoral rejection, a vexing outcome of organ transplantation, has been attributed by some to activation and by others to apoptosis of endothelial cells in the graft. We asked which of these processes causes acute vascular rejection by tracing the processes during the development of acute vascular rejection in porcine cardiac xenografts performed in baboons. Apoptosis, assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), expression of activated caspase-3, and proapoptotic genes Bax and Bcl-x L , was not detected until acute vascular rejection was well advanced, and even then, apoptosis was largely confined to myocytes. Activation of the endothelium, as evidenced by expansion of rough endoplasmic reticulum and increased ribosomal antigen and phospho-p70 S6 kinase, occurred early in the course of acute vascular rejection and progressed through the disease process. These findings suggest that acute vascular rejection is caused by an active metabolic process and not by apoptosis in the endothelium.