Modulation of FLT3 signal transduction through cytoplasmic cysteine residues indicates the potential for redox regulation.

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.

Polyunsaturated liposomes are antiviral against hepatitis B and C viruses and HIV by decreasing cholesterol levels in infected cells.

The pressing need for broad-spectrum antivirals could be met by targeting host rather than viral processes. Cholesterol biosynthesis within the infected cell is one promising target for a large number of viral systems, including hepatitis C virus (HCV), hepatitis B virus (HBV) and HIV. Liposomes developed for intracellular, endoplasmic reticulum (ER)-targeted in vivo drug delivery have been modified to include polyunsaturated fatty acids that exert an independent antiviral activity through the reduction of cellular cholesterol. These polyunsaturated ER liposomes (PERLs) have greater activity than lovastatin (Mevacor, Altoprev), which is clinically approved for lowering cholesterol and preventing cardiovascular disease. Treatment of HCV, HBV, and HIV infections with PERLs significantly decreased viral secretion and infectivity, and pretreatment of naïve cells reduced the ability of both HCV and HIV to establish infections because of the decreased levels of plasma membrane cholesterol. Direct competition for cellular receptors was an added effect of PERLs against HCV infections. The greatest antiviral activity in all three systems was the inhibition of viral infectivity through the reduction of virus-associated cholesterol. Our study demonstrates that PERLs are a broadly effective antiviral therapy and should be developed further in combination with encapsulated drug mixtures for enhanced in vivo efficacy.

Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases.

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 RPTPalpha 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.

Visualization of SHP-1-target interaction.

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.