Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTP␣ promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.Cellular receptors for growth factors, hormones, cytokines, and antigens are postranslationally modified with N-linked, branched carbohydrate chains. Nascent polypeptide chains become initially glycosylated with a mannose-rich branched oligosaccharide in the endoplasmic reticulum (ER). Then, the glycoproteins are subjected to partial deglycosylation by several selective glycosidases, eventually enabling transfer to the Golgi compartment and more complex glycosylation (9). This process, designated glycoprotein maturation, is coupled to stringent quality control in the ER (4, 10). Correct folding is monitored by a complex system comprising, among other components, the chaperones calnexin and calreticulin, the oxidoreductase ERp57, and the glycosylation enzymes UDP-glucose glucosyltransferase and glucosidases I and II. Improperly folded glycoproteins are tagged by reversible glucosylation, enabling their interactions with calnexin and calreticulin and leading to their retention in the ER (4). Properly folded glycoproteins can dissociate from the chaperones and proceed to the Golgi compartment for further glycosylation.The receptor tyrosine kinase (RTK) Fms-like tyrosine kinase 3 (FLT-3) is expressed in multiple hematopoietic lineages (21,22). Constitutively active FLT-3 mutants, notably, versions harboring internal tandem duplications in the juxtamembrane domain (FLT-3 ITD) and versions with point mutations in the kinase activation loop, have been found in approximately ...

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273,355 and 457 nm and the isoelectric point is at pH 6.2.The K,,, value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the K,,, for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag' and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure. . Even if the biological importance of the more specific L-amino acid oxidases is still unknown, they can serve as valuable analytical tools for the determination of L-amino acids. In particular, the specific determination of Lglutanlate would provide new possibilities for the estimation of serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in the clinical laboratory and is also of interest for evaluating the quality of foods.The hitherto described L-glutamate oxidases from Streptomycetes catalyze the oxidation of L-glutamic acid to 2-oxoglutaric acid, simultaneously generating NH3 and H 2 0 2 . However, the enzyme from S. violuscens oxidizes Lglutamine and L-histidine [13] in addition to L-glutamate and the enzyme from S. X-119-6 oxidizes L-aspartic acid [14] to some extent.In this paper we report on the isolation, purification and characterization of a novel L-glutamate oxidase from the culture filtrate of Streptomyces endus, which is entirely specific for the conversion of L-glutamic acid.

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion.

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Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with ‘internal tandem duplications (FLT3ITD)’ drive production of reactive oxygen in leukemic cells.FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions.The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein.

Signaling of receptor tyrosine kinases (RTKs) is regulated by protein-tyrosine phosphatases (PTPs). We previously discovered the efficient downregulation of Ros RTK signaling by the SH2 domain PTP SHP-1, which involves a direct interaction of both molecules. Here, we studied the mechanism of this interaction in detail. Phosphopeptides representing the SHP-1 candidate binding sites in the Ros cytoplasmic domain, pY2267 and pY2327, display high affinity binding to the SHP-1 N-terminal SH2 domain (Kd=217 nM and 171 nM, respectively). Y2327 is, however, a poor substrate of Ros kinase and, therefore, contributes little to SHP-1 binding in vitro. To explore the mechanism of association in intact cells, functional fluorescent fusion proteins of Ros and SHP-1 were generated. Complexes of both molecules could be detected by Förster resonance energy transfer (FRET) in intact HEK293 and COS7 cells. As expected, the association required the functional SHP-1 N-terminal SH2 domain. Unexpectedly, pY2267 and pY2327 both contributed to the association. Mutation of Y2327 reduced constitutive association in COS7 cells. Ligand-dependent association was abrogated upon mutation of Y2267 but remained intact when Y2327 was mutated. A phosphopeptide representing the binding site pY2267 was a poor substrate for SHP-1, whereas Ros activation loop phosphotyrosines were effectively dephosphorylated. We propose a model for SHP-1-Ros interaction in which ligand-stimulated phosphorylation of Ros Y2267 by Ros, phosphorylation of Y2327 by a heterologous kinase, and inactivation of Ros by SHP-1-mediated dephosphorylation play a role in the regulation of complex stability.

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