In nonribosomal biosynthesis of peptide antibiotics by multimodular synthetases, amino acid monomers are activated by the adenylation domains of the synthetase and loaded onto the adjacent carrier protein domains as thioesters, then the formation of peptide bonds and translocation of the growing chain are effected by the synthetase's condensation domains. Whether the condensation domains have any editing function has been unknown. Synthesis of aminoacyl-coenzyme A (CoA) molecules and direct enzymatic transfer of aminoacyl-phosphopantetheine to the carrier domains allow the adenylation domain editing function to be bypassed. This method was used to demonstrate that the first condensation domain of tyrocidine synthetase shows low selectivity at the donor residue (D-phenylalanine) and higher selectivity at the acceptor residue (L-proline) in the formation of the chain-initiating D-Phe-L-Pro dipeptidyl-enzyme intermediate.

Calcineurin, a Ca2+, calmodulin-dependent protein phosphatase, was recently found to bind with high affinity to two different immunosuppressant binding proteins (immunophilins) with absolute dependence on the presence of the immunosuppressants FK506 or cyclosporin A (CsA) [Liu et al. (1991) Cell 66, 807-815]. The binding affinities of the immunophilin-drug complexes toward calcineurin and the stoichiometry of the resultant multimeric complexes have now been determined, and structural elements of FK506, CsA, and calcineurin that are critical for mediating their interactions have been identified. Analogues of FK506 (FK520, FK523, 15-O-demethyl-FK520) and CsA (MeBm2t1-CsA and MeAla6-CsA) whose affinities for their cognate immunophilins do not correlate with their immunosuppressive activities have been prepared and evaluated in biochemical and cellular assays. We demonstrate a strong correlation between the ability of these analogues, when bound to their immunophilins, to inhibit the phosphatase activity of calcineurin and their ability to inhibit transcriptional activation by NF-AT, a T cell specific transcription factor that regulates IL-2 gene synthesis in human T cells. In addition, FKBP-FK506 and CyP-CsA do not inhibit members of the PP1, PP2A, and PP2C classes of serine/threonine phosphatases. These data suggest that calcineurin is the relevant cellular target of these immunosuppressive agents and is involved in Ca(2+)-dependent signal transduction pathways in, among others, T cells and mast cells.

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Extracellular growth and differentiation factors induce changes in gene expression in the nucleus by initiating a series of protein associations that alter the subcellular localization of intracellular signaling proteins. Initial events involve receptor homo-or heterodimerization and subsequent recruitment of cytosolic signaling proteins to the inner leaflet of the plasma membrane. Intermediate events involve the translocation of proteins into the nucleus. Late events involve the recruitment of transcriptional activators to the vicinity of specific genes in the nucleus, resulting in increased gene transcription. MATERIALS AND METHODSDNA Constructs. All expression constructs are in the vector pBJ5 (2) and were created through ligating fragments generated by oligonucleotide synthesis or PCR into pBJ5 or derivatives thereof. All fragments generated by PCR were sequenced. The following abbreviations are used to describe the expression constructs: M, myristoylation sequence from c-Src (residues 1-14); Fpk, human FKBP12 with the mutations G89P and 19OK; C, murine cyclophilin C (residues 36-212); F, intracellular domain of human Fas receptor (residues 179-319); G, DNA binding domain of GAL4 (residues 1-147); J, Aequorea victoria green fluorescent protein with the fluorescence-enhancing mutation S65T; V, activation domain of VP16 (residues 413-490); E, hemagglutinin epitope tag; E', FLAG epitope tag. The number of repeats of a given fragment is indicated (if in multiples) following the fragment's designation.Cell 250 V, 800 ,uF, 129 Qi 4-mm cuvettes in a total volume of 220 ,ul of RPMI 1640 medium)] with 10-min incubations at room temperature before and after electroporation. COS-7 cells (106 cells) were electroporated (BTX model 600 electroporator; 180 V, 1000 ,tF, 72 fl 2-mm cuvettes in a total volume of 400 p,l of phosphate buffered saline) with 5-min incubations on ice before and after electroporation.