(22), and Arabidopsis thaliana (23), mutants with defects in RBPs are defective in cell growth and differentiation. An example of an RBP that regulates development is provided by the Bruno protein and its role as a translational repressor of oskar mRNA. In Drosophila, oskar is required for formation of germ cells and positioning of the posterior of the embryo (24). Both oskar mRNA and the encoded protein must be properly localized to the posterior pole of the oocyte for correct development (25,26). Localized expression of Oskar protein is determined in part by translational silencing of the oskar mRNA outside of the posterior of the oocyte. This repression is mediated by cis-acting sequences in the 3Ј-untranslated region (UTR) of oskar mRNA called Bruno response elements (BREs) and a corresponding trans-acting factor, the Bruno protein. Deletion of these BREs results in inappropriate translation of Oskar protein in the anterior end of the oocyte leading to embryos with two posterior poles. The Bruno protein is an RRM-containing protein present in oocytes. Extracts prepared from Drosophila ovaries recapitulate this Bruno-dependent translational repression of oskar mRNA in vitro (27). By regulating the localized expression of Oskar, Bruno has a key role in germ cell formation and early embryogenesis.The Bruno protein is similar in domain structure to the Elav family of proteins (28). Members of this family provide exam-
Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47phox, during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47phox. Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47phox and Akt was shown by the ability of GST-p47phox to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47phox in vitro, as shown by 32P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser304 and Ser328, were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47phox phosphorylation, which contributes to respiratory burst activity in human neutrophils.
Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3 and endogenous 14-3-3 from HEK293 cell lysates. Recombinant active PKB/ Akt phosphorylated recombinant 14-3-3 in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3. Based on a motif search of 14-3-3, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3 with recombinant active PKB/ Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3 dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3 both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.