Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the a and jB subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3a (Statll3, Stat84, and Stat9l), and the pl35'yk2 and Jak-1 tyrosine kinases. In this report, we demonstrate that the a subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the pl35cyk2 tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the a subunit can bind the pl35'yk2 contained in human cell lysates. The association between the a subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, pl35tk2, but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFNa-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the a subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs.
PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, β, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, β, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.
The issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2~SH2! domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85~including just the two SH2 domains and the inter-SH2 region!, both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein.
In the normal adult brain, glucose provides 90% of the energy requirements as well as substrate for nucleic acid and lipid synthesis. In the present study, effects of ethanol on glucose transporters (GLUT) and glucose utilization were examined in rat brain. Male Sprague-Dawley rats weighing 250-300 gms were given either ethanol 3 gm/kg BW or saline i.p. 4 hrs prior to the animal sacrifice and removal of the cerebral cortical tissue. The cortical plasma membranes analyzed by cytochalasin B binding assay showed a decrease in GLUT number but not in GLUT affinity in the ethanol treated rats as compared to the control rats. The estimated Ro values were 70 +/- 8.9 Vs 91 +/- 8.9 pmoles/mg protein (p < 0.05 N=4) and the estimated Kd values were 0.37 +/- 0.03 and 0.28 +/- 0.05 microM (p: NS) in ethanol and control experiments respectively. Immunoblots of purified cerebral plasma membranes and low density microsomal fraction showed 17% and 71% decrease for GLUTI and 54% and 21% (p<0.05 or less; n=6) for GLUT3 respectively in ethanol treated rats than in control animals. Immunofluoresence studies also showed reduction of GLUT1 immunoreactively in choroid plexus and cortical microvessels of ethanol treated rats as compared to control rats. The effect of ethanol on regional cerebral metabolic rates for glucose (CMR(Glc)) was studied using [6-(14)C] glucose and showed statistically insignificant decrease in brain glucose utilization. These data suggest that ethanol in-vivo decrease GLUT number and protein content in rat cerebral cortex.
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