Protein kinase A-dependent phosphorylation modulates beta1Pix guanine nucleotide exchange factor activity through 14-3-3beta binding.
beta(1)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 betaPix and endogenous 14-3-3beta was increased after forskolin stimulation and significantly inhibited by protein kinase A inhibitor. However, forskolin stimulation failed to increase the interaction between 14-3-3beta and a beta(1)Pix mutant that is insensitive to protein kinase A phosphorylation, beta(1)Pix(S516A, T526A). We present evidence indicating that forskolin-induced binding of 14-3-3beta to beta(1)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 beta(1)Pix homodimerization and 14-3-3beta binding and modulates beta(1)Pix-GEF activity. These residues also play a crucial role in beta(1)Pix intracellular localization. These results indicate that 14-3-3beta negatively affects the GEF activity of dimeric beta(1)Pix only. Altogether, these results provide a mechanistic insight into the role of 14-3-3beta in modulating beta(1)Pix-GEF activity.
Chahdi
Ahmed
A
Division of Nephrology and Kidney Disease Center, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. sorokin@mcw.edu
Sorokin
Andrey
A
D048948:14-3-3 Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019281:Dimerization; D018274:Electroporation; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008954:Models, Biological; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D020033:Protein Isoforms; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D005576:Colforsin; D013912:Threonine; D012694:Serine; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D020830:rac1 GTP-Binding Protein