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The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.Cyclosporin A (CsA), FK506, and rapamycin are potent immunosuppressive drugs that inhibit T-lymphocyte proliferation. The action of these drugs is mediated by intracellular receptors, termed immunophilins, that bind either CsA (cyclophilins) or FK506 and rapamycin (FK506-binding proteins [FKBPs]) (for reviews, see references 43 and 47). Previous studies identified these receptors as abundant, cytosolic proteins possessing an inherent peptidyl prolyl cis-trans isomerase (PPIase; rotamase) activity that is inhibited by drug ligand binding (12,16,46).The first FKBP to be described in detail at the protein (16, 46) and cDNA (25, 53) levels was FKBP12, a ubiquitous immunophilin of 11.8 kDa highly conserved in eukaryotes. More recently, additional members of this family have been identified on the basis of their ability to bind FK506 and rapamycin. Sequence analysis of the cloned genes corresponding to FKBP12.6, FKBP13, FKBP25, and FKBP52 (named to reflect their molecular weights) from mammalian sources reveals extensive conservation of amino acid sequence, in particular in the protein domain responsible for drug ligand binding and PPIase activity (for reviews, see references 15 and 58).Although the actions of FK506 and rapamycin are mediated by the same family of intracellular receptors, these drugs achieve immunosuppression by different mechanisms (5). Rapamycin inhibits a calcium-independent event involved in the proliferative response of T cells to growth ...

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Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a XcDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.Glucocorticoids induce cytolysis in susceptible lymphocyte populations, including immature thymocytes and certain leukemias and lymphomas. Steroid-induced cell death is thought to occur through the activation of an endogenous suicide process. Glucocorticoid treatment of murine lymphocytes results in severe alterations in cellular metabolism which include an inhibition of glucose transport (50), increased RNA (14) and protein (44) degradation, a rise in intracellular calcium levels (37), and decreased incorporation of thymidine into DNA (9). Glucocorticoid-induced lymphocytolysis is preceded by cell cycle arrest (28) and extreme morphological changes. These include widespread chromatin condensation, which is associated with extensive DNA fragmentation in both human and murine lymphocytes (22, 58). The DNA cleavage appears to result from the activation of a preexisting calcium-dependent endonuclease (17, 59). Glucocorticoid-induced DNA fragmentation and cell death are prevented when RNA and protein syntheses are inhibited (17). In addition, there is genetic evidence in mouse and human cells for the existence of a locus involved in cell lysis (25,26,63 (21,32,53,62). The possibility thus exists that both glucocorticoids and cAMP regulate the expression of genes involved in the lytic process.Glucocorticoids and cAMP are known to regulate the levels of several proteins in lymphocytes. For example, glucocorticoids induce glucocortin in rat thymus (18), glutamine synthetase in th...

Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.

Gene VI of the cauliflower mosaic virus

The Lii ribosomal protein operon in Escherichia coli consists of the genes for proteins Lii and Li and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the translation initiation site of the first Lii cistron. Several mutant plasmid molecules carrying altered nucleotide sequences in the Li target site were constructed by site-directed in vitro mutagenesis using synthetic oligodeoxyribonucleotides. Specifically, we examined the importance of a presumptive double-stranded stem structure that is common among Li binding sites on rRNA from a variety of organisms and in LI1 mRNA. Mutational alterations that disrupt the stem structure were found to abolish translational regulation as analyzed both in vitro and in vivo. Two of the mutations were combined so that the stem structure was restored but with a different primary nucleotide sequence. This double mutant was shown to restore the original phenotype, the ability to be translationally regulated by L1. These experiments show the importance of the stem structure, but not its primary sequence, for the interaction of Li with the mRNA and support the concept that mRNA target sites share some structural features with the corresponding ribosomal protein binding sites of rRNA. MATERIALS AND METHODS Oligodeoxyribonucleotides used for mutagenesis were prepared by the phosphotriester synthesis method (10). dT-T-A-T-T-A-C-G-G-A-A-C-T-T-G-A-G was synthesized in thislaboratory and purified by polyacrylamide gel electrophoresis. Other oligonucleotides (see Fig. 3) were synthesized by Bruce Kaplan (City of Hope Research Institute) and were purified by HPLC. Mutagenesis directed with these oligonucleotides was carried out essentially as described by Zoller and Smith (1i). DNA sequence determination of mutant plasmids was carried out by the method of Maxam-Gilbert-.(12) from the HindIII site in the EcoRI fragment used as the target DNA for mutagenesis (see Fig. 2). Standard techniques were used for construction of various plasmids and M13 phage derivatives (13,14).DNA-dependent in vitro protein synthesis was carried out as described (2, 15). Protein Li, purified previously (16), was stored at -70'C and used for the in vitro experiments as described (2,6). Measurements of relative synthesis rates of r proteins in vivo were carried out as described in the legend to 5389The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.