Open AccessProtein Kinase A-Dependent Phosphorylation Modulates β1Pix Guanine Nucleotide Exchange Factor Activity through 14-3-3β Binding
Author(s) -
Ahmed Chahdi,
Andrey Sorokin
Publication year - 2008
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.00898-07
Subject(s) - guanine nucleotide exchange factor , biology , forskolin , gtp' , gtpase , phosphorylation , protein kinase a , microbiology and biotechnology , leucine zipper , cdc42 , biochemistry , gtp binding protein regulators , kinase , g protein , signal transduction , peptide sequence , in vitro , enzyme , gene
β1 Pix is a guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42 which has been shown to mediate signaling pathways leading to cytoskeletal reorganization. In the present study, we show that the basal association between endogenous βPix and endogenous 14-3-3β was increased after forskolin stimulation and significantly inhibited by protein kinase A inhibitor. However, forskolin stimulation failed to increase the interaction between 14-3-3β and a β1 Pix mutant that is insensitive to protein kinase A phosphorylation, β1 Pix(S516A, T526A). We present evidence indicating that forskolin-induced binding of 14-3-3β to β1 Pix results in inhibition of Rac1 GTP loading in 293 cells and in vitro. Furthermore, we show that deletion of 10 amino acid residues within the leucine zipper domain is sufficient to block β1 Pix homodimerization and 14-3-3β binding and modulates β1 Pix-GEF activity. These residues also play a crucial role in β1 Pix intracellular localization. These results indicate that 14-3-3β negatively affects the GEF activity of dimeric β1 Pix only. Altogether, these results provide a mechanistic insight into the role of 14-3-3β in modulating β1 Pix-GEF activity.