PubMed · 9095189
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J T Nguyen, W A Lim. 1997. How Src exercises self-restraint.. https://doi.org/10.1038/nsb0497-256
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Selective protein kinase C (PKC) activators and inhibitors and a physiological agonist, fMLP, were used to study superoxide production and PKC isoenzyme activation in human neutrophils. The data show that the classical PKC isoenzymes, alpha and beta, were activated by TPA and at a time prior to NADPH oxidase complex assembly. fMLP induced activation of PKC-beta over a similar time course. Inhibition of c-PKCs reduced, but did not block, TPA-induced superoxide production completely, suggesting additional PKC isoenzymes were involved beyond NADPH oxidase assembly. PKC inhibitors were unable to inhibit fMLP-induced superoxide generation, indicative of signal redundancy in the induction of superoxide generation in human neutrophils.
Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done. G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4. The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.
The crystal structure of the protein tyrosine phosphatase SHP-2 reveals the mechanism of auto-inhibition of phosphatase activity by its SH2 domains. Phosphotyrosine peptide stimulation of the phosphatase activity, resulting from peptide binding to the N-terminal SH2 domain, is linked to conformational changes within the protein, including an unprecedented allosteric transition of the N-terminal SH2 domain.