The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.Receptor protein-tyrosine kinases (RPTKs) 1 regulate key signal transduction cascades that control cellular growth and proliferation. The stem cell factor (SCF) receptor c-Kit is a type III transmembrane RPTK comprised of five extracellular immunoglobulin domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (1, 2). The type III RPTK family includes c-Kit (3), the colonystimulating factor-1 (formerly FMS) (4), the platelet-derived growth factor ␣ and ␤ receptors (1, 5), and the FMS-related receptor FLT-3 (6). Signaling by RPTKs occurs via ligand binding to the extracellular IG domains, inducing the receptors to form dimers, and thereby activating intrinsic tyrosine kinase activity through the transphosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (7,8). Ligand binding both activates kinase activity and creates tyrosine-phosphorylated receptors that mediate the specific binding of intracellular signaling proteins. Src homology 2 and protein tyrosine binding domains (9), including the proteintyrosine phosphatase SHP-1, act as negative regulators of c-Kit activity (10). These cytoplasmic signaling proteins initiate serine/threonine phosphorylation cascades that activate transcription factors to determine specific cellular responses (Fig. 1).The human c-Kit gene is the cellular homologue of the v-kit oncogene found in the transforming Hardy-Zuckerman 4 feline sarcoma virus (11) and encodes a 976-amino acid residue RPTK. Loss-of-function c-Kit mutations establish its importance for the normal growth of hematopoietic progenitor cells, mast cells, melanocytes, primordial germ cells, and the interstitial cells of Cajal (12-15). Gain-of-function mutations, resulting in SCF-independent, constitutive activation of c-Kit, are found in several highly malignant cancers. Mutations in the c-Kit juxtamembrane region cluster around the two main autophosphorylation sites that mediate protein tyrosine binding, Tyr-568 and Tyr-570, and are associated with human gastrointestinal stromal t...

Fibroblast activation protein ␣ (FAP␣) is highly expressed in epithelial cancers and has been implicated in extracellular matrix remodeling, tumor growth, and metastasis. We present the first high resolution structure for the apoenzyme as well as kinetic data toward small dipeptide substrates.

Microsomal cytochrome P450s participate in xenobiotic detoxification, procarcinogen activation, and steroid hormone synthesis. The first structure of a mammalian microsomal P450 suggests that the association of P450s with the endoplasmic reticulum involves a hydrophobic surface of the protein formed by noncontiguous portions of the polypeptide chain. This interaction places the entrance of the putative substrate access channel in or near the membrane and orients the face of the protein proximal to the heme cofactor perpendicular to the plane of the membrane for interaction with the P450 reductase. This structure offers a template for modeling other mammalian P450s and should aid drug discovery and the prediction of drug-drug interactions.

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We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c 552 gene. Key features are~1! construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence~MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla! followed by the amino acid sequence of mature Thermus cytochrome c 552 ; and~2! coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes ccmABCDEFGH !. Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC 552 , and native cytochrome c 552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba 3 , a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1 H-NMR spectroscopies. The 1.7 Å resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein~Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644!. This approach may be generally useful for expression of alien cytochrome c genes in E. coli. Keywords: cytochrome c; Escherichia coli; homologous expression; Thermus thermophilusAlthough studied for many decades as part of the cell's respiratory apparatus~Lemberg & Barrett, 1973;Mathews, 1985;Pettigrew & Moore, 1987!, cytochromes c remain of considerable interest as objects in the study of electron transfer reactions~Ferguson-Miller et al., 1979;Pan et al., 1993;Bjerrum et al., 1995;Geren et al., 1995;Winkler et al., 1995! and of protein folding~Sosnick et al., 1994;Bryngelson et al., 1995;Mines et al., 1996;Bai, 1999!. In addition, recent evidence indicates that cytochrome c released from the mitochondrion is able to initiate apoptosis~Wallace, 1999, and references therein!, suggesting that this protein may have functions other than electron transfer. Today, application of modern experimental approaches to cytochrome c function can be limited by the general unavailability of suitable expression systems for cloned cytochrome c genes. Escherichia coli is certainly the organism of choice, but most attempts to express foreign cytochrome c genes in this bacterium have been unsuccessful; either the yield is imprac-