The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1͞FRAP͞mTOR, the in vivo rapamycin-sensitive target and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. Here, we show that RAFT1 directly phosphorylates p70 S6k , 4E-BP1, and 4E-BP2 and that serum stimulates RAFT1 kinase activity with kinetics similar to those of p70 S6k and 4E-BP1 phosphorylation. RAFT1 phosphorylates p70 S6k on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1-eIF-4E interaction in vivo. RAFT1 phosphorylates p70 S6k much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. This raises the possibility that, in vivo, an unidentified kinase analogous to p70 S6k is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1.Increases in the translation of certain mRNAs are an important response to mitogen stimulation (1-3), but little is known about the signaling pathways that link growth stimuli to the activation of the protein synthesis machinery. Studies with rapamycin (4), a potent immunosuppressant, have uncovered a signaling pathway that modulates protein synthesis in yeast (5) and animal cells (6, 7). In vivo treatment with rapamycin affects the phosphorylation of several regulators of translation, including the ribosomal S6 protein and its specific kinase, p70 S6k (8, 9); the eIF-4E binding proteins, 4E-BP1 (6, 7) and 4E-BP2 (10); and elongation factor 2 (11).Ribosomal S6 and 4E-BP1 regulate the initiation of translation of distinct classes of mRNAs. Phosphorylation by p70 S6k of the S6 protein of the small ribosomal subunit permits, through unknown mechanisms, efficient translation of mRNAs containing oligopyrimidine tracts in their 5Ј untranslated regions (12, 13). Phosphorylation of 4E-BP1 controls capdependent translation of mRNAs with extensive secondary structure (3). Initiation factor 4F complexes with these mRNAs through the interaction of its eIF-4G subunit with eIF-4E, the cap-binding protein that recognizes the N 7 -methyl-GpppN structure of the 5Ј end of all nonorganellar mRNAs. In quiescent cells, 4E-BP1 competes with eIF-4G for binding to eIF-4E and represses translation by displacing the initiation factor 4F from the mRNA. Growth stimuli activate phosphorylation of 4E-BP1, which decreases its affinity for eIF-4E and releases the block on cap-dependent translation (3). By preventing the phosphorylation of specific residues on p70 S6k and 4E-BP1, rapamycin inhib...

Calcineurin is a widely distributed protein phosphatase regulated by calcium and calmodulin. It mediates the immunosuppressive actions of drugs such as cyclosporin and FK506, and has been implicated in a number of calcium-sensitive pathways in the nervous system, including regulation of neurotransmitter release and modulation of long-term changes in synaptic plasticity. Calcineurin associates physiologically with other proteins, including calmodulin, FKBP12 (FK506-binding protein), the ryanodine receptor, and the inositol 1,4,5-trisphosphate receptor. We now report the identification, molecular cloning, and functional characterization of a novel protein, cain (calcineurin inhibitor), that interacts with and inhibits calcineurin. The full-length cain cDNA predicts a 240-kDa protein with no significant homology to any known protein. Cain associates with calcineurin both in vitro and in vivo, leading to a non-competitive inhibition of calcineurin activity. The putative calcineurin-binding domain of cain, a 38-amino acid region defined by mutational analysis, is highly basic. Like calcineurin, cain has a prominent neuronal expression and a wide tissue distribution. Cain's expression pattern in the brain closely resembles that of calcineurin, indicating a physiologic association between the two proteins.

Epithelial-cadherin (E-cadherin) is a master organizer of the epithelial phenotype. Its function is regulated in part by p120-catenin (referred to herein as p120), a cytoplasmic binding partner that directly regulates cadherin stability. As it has been suggested that cadherins have a role in inflammatory bowel disease (IBD), we sought to investigate this further by assessing the effect of p120 deficiency in mouse small intestine and colon. p120 conditional KO mice were superficially normal at birth but declined rapidly and died within 21 days. Cellcell adhesion defects and inflammation led to progressive mucosal erosion and terminal bleeding, similar to what is observed in a dominant-negative cadherin mouse model of IBD. Additionally, selective loss of adherens junctions and accumulation of atypical COX-2-expressing neutrophils in p120-null areas of the colon were observed. To elucidate the mechanism, direct effects of p120 deficiency were assessed in vitro in a polarizing colon cancer cell line. Notably, transepithelial electrical resistance was dramatically reduced, neutrophil binding was increased 30 fold, and levels of COX-2, an enzyme associated with IBD, were markedly increased in neutrophils. Our data suggest that p120 loss disrupts the neonatal intestinal barrier and amplifies neutrophil engagement and that these changes lead to catastrophic inflammation during colonization of the neonatal gut with bacteria and other luminal antigens. Thus, we conclude that p120 has an essential role in barrier function and epithelial homeostasis and survival in the intestine.

The first important insight into how immunosuppres-School of Medicine sants act came with the observation by Handschumacher Johns Hopkins University and associates (1984) that CsA binds to a cytoplasmic Baltimore, Maryland 21205 18 kDa protein designated cyclophilin A. Subsequently,

RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.

Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC 50 for calcium in the range of 0.1-0.4 M. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calciumsensing mechanism that facilitates endocytosis.Neurotransmitter release occurs by calcium-dependent exocytosis of synaptic vesicles from nerve terminals mediated by a complex of proteins whose interactions are calcium-dependent (1, 2). Endocytic recycling of released synaptic vesicles is initiated at the same time as exocytosis and involves a complex of endocytic proteins including clathrin, clathrin adapters, dynamin 1, amphiphysin, and synaptojanin (3-5). Dephosphorylation of endocytic proteins is required for their assembly into a functional complex (6). A possible link between calcium and dephosphorylation is suggested by evidence that the calciumsensitive phosphatase calcineurin (Cn) 1 can dephosphorylate endocytic proteins (7-9) and that the drugs cyclosporin A and FK506, which inhibit Cn, impair endocytosis (10, 11). However, the evidence for calcium-dependence in endocytosis has been conflicting (11-17). We now provide direct evidence that Cn is physically linked to the endocytic machinery. We show that Cn binds to dynamin 1 independent of its catalytic activity and that the Cn-dynamin 1 complex combines with amphiphysin 1, the anchor protein of the endocytic complex. Moreover, we demonstrate that the Cn-dynamin 1 interaction is calcium-dependent, allowing this complex to act as a calcium sensor. Finally, we show that disruption of the Cn-dynamin 1 interaction leads to inhibition of clathrin-mediated endocytosis. EXPERIMENTAL PROCEDURESAffinity Purification of the Cn-Dynamin 1 Complex-Adult rat brain was homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 0.2% Triton X-100, 0.5 mM ␤-mercaptoethanol, 5 g/ml aprotinin, 1 g/ml leupeptin, 6 g/ml chymostatin, 0.7 g/ml pepstatin, 1 mM PMSF) and centrifuged at 20,000 ϫ g for 20 min to remove insoluble materials. The resulting lysate was divided into equal aliquots and incubated with GST-FKBP12-Sepharose beads for 2 h at 4°C. Where indicated, GST-FKBP12-Sepharose beads were preabsorbed with FK506 for 1 h at 4°C and then washed twice in lysis buffer prior to adding to the cell lysate. Following the incubation, the Sepharose beads were washed three times with lysis buffer and boiled in SDS sample buffer. Eluted proteins were separated by SDS-PAGE and stained with Coomassie Blue or tra...

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The multitargeted tyrosine-kinase inhibitor sunitinib has emerged as one of the standards of care for goodand intermediate-risk metastatic renal cell carcinoma.Although generally associated with acceptable toxicity, sunitinib exhibits a novel and distinct toxicity profile that requires monitoring and management. Fatigue, di-