Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2′,3,4,4′-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.
The concentration dependence of the unidirectional chloride flux and the inhibition of the unidirectional chloride flux by sulfate were studied in human red cell ghosts. The concentration dependence of the unidirectional chloride flux and its inhibition by sulfate were asymmetric. The unidirectional chloride flux can be saturated from the inner and from the outer membrane surface. For the inner membrane surface, lower chloride half-saturation constants were obtained than for the outer membrane surface. The inhibition of the unidirectional chloride flux by sulfate is competitive. In contrast to the chloride half-saturation constants, the inhibition constants of sulfate for the inner membrane surface were higher than the inhibition constants of sulfate for the outher membrane surface. Either there are fixed anion binding sites at the inner and at the outer membrane surface which control the access of anions to a pore, or there is a mobile carrier which is in contact with both membrane surfaces. The asymmetry of the concentration response and of the inhibition of the unidirectional chloride flux suggest that the anion binding sites at the inner and at the outer membrane surface differ with respect to their affinities for chloride and for sulfate. Alternatively, the asymmetry of the chloride transport system could indicate an asymmetric distribution of a mobile anion carrier across the erythrocyte membrane.
The concentration dependence of the sulfate and the phosphate selfexchange and homoexchange fluxes was studied in resealed red cell ghosts (25 degrees C, pH 7.3). The selfexchange fluxes were calculated from the rate constant of the tracer back-exchange and from the intracellular substrate anion content. The homoexchange fluxes were determined from the initial cis-to-trans tracer fluxes and the initial specific substrate anion activities at the cis-membrane side. Sulfate and phosphate concentrations ranging from approx. 2-100 mM were employed. The selfexchange fluxes of sulfate and of phosphate exhibit sigmoidal flux/concentration curves. The apparent Hill coefficients were in the range of 1.2-1.4 indicating a type of positive cooperativity. Under homoexchange conditions the positive cooperativity of the flux/concentration curves disappears. The outward homoexchange fluxes of sulfate and phosphate display a saturation kinetics with Hill coefficients close to 1.0. The inward homoexchange fluxes exhibit a negative type of cooperativity with Hill coefficients smaller than 1.0. The sulfate and the phosphate half-saturation concentrations for the outer and the inner membrane surface are equal in size and amount to approx. 35 mM for sulfate and to approx. 110 mM for phosphate, respectively. The positive cooperativity of the unidirectional sulfate and phosphate fluxes under selfexchange conditions and the disappearance of the positive cooperativity under homoexchange conditions indicate a cooperativity of the translocation process. The saturation of the outward homoexchange flux and the negative cooperativity of the inward homoexchange flux suggest a substrate anion binding according to the law of mass action at the inner and a negative cooperativity of substrate anion binding at the outer membrane surface.
The concentration dependence and the pH dependence of the phosphate transport across the red cell membrane were investigated. The unidirectional phosphate fluxes were determined by measuring the 32P-phosphate self-exchange in amphotericin B (5 mumol/liter) treated erythrocytes at 25 degrees C. The flux/concentration curves display an S-shaped increase at low phosphate concentrations, a concentration optimum in the range of 150 to 200 mM phosphate and a self-inhibition at high phosphate concentrations. The apparent half-saturation concentrations, P(0.5), range from 50 to 70 mM and are little affected by pH. The self-inhibition constants, as far as they can be estimated, range from 400 to 600 mM. The observed maximal phosphate fluxes exhibit a strong pH dependence. At pH 7.2, the actual maximal flux is 2.1 X 10(-6) moles . min-1 . g cells-1. The ascending branches of the flux/concentration curves were fitted to the Hill equation. The apparent Hill coefficients were always in the range of 1.5-2.0. The descending branches of the flux/concentration curves appear to follow the same pattern of concentration response. The flux/pH curves were bell-shaped and symmetric with regard to their pH dependence. The pH optimum is at approximately pH 6.5-6.7. The apparent pK of the activator site is in the range of 7.0 to 7.2, while the apparent pK for the inactivating site is in the range of 6.2 to 6.5. The pK-values were not appreciably affected by the phosphate concentration. According to our studies, the transport system possesses two transport sites and probably two modifier sites as indicated by the apparent Hill coefficients. In addition, the transport system has two proton binding sites, one with a higher pK that activates and one with a lower pK that inactivates the transport system. Since our experiments were executed under self-exchange conditions, they do not provide any information concerning the location of these sites at the membrane surfaces.
Mutations in the PKD2 gene lead to the development of polycystic kidney disease (PKD). The PKD2 gene codes for polycystin-2, a cation channel with unknown function. The cytoplasmic, C-terminal domain interacts with a large number of proteins including mDia1, alpha-actinin, PIGEA-14, troponin, and tropomyosin. The C-terminal fragment polycystin-2 (680-796) consisting of 117 amino acids contains a putative calcium binding EF-hand. It was produced in Escherichia coli and enriched uniformly with (13)C and (15)N. The backbone and side chain resonances were assigned by multidimensional NMR methods, the obtained chemical shifts are typical for a partially folded protein. The chemical shifts obtained are in line with the existence of two paired helix-loop-helix (HLH) motifs.
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