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Leucine zipper-mediated homo-oligomerization regulates the Rho-GEF activity of AKAP-Lbc.
Journal of Biological Chemistry
Date de publication
Publication types: Journal Article
AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha-subunit of the heterotrimeric G protein G(12), whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family.
14-3-3 Proteins/chemistry, A Kinase Anchor Proteins, Adaptor Proteins, Signal Transducing/chemistry, Amino Acid Motifs, Blotting, Western, Cell Line, Electrophoresis, Polyacrylamide Gel, GTP-Binding Protein alpha Subunits, G12-G13/chemistry, Genetic Vectors, Glutathione Transferase/metabolism, Green Fluorescent Proteins/metabolism, Guanine Nucleotide Exchange Factors/chemistry, Humans, Immunoprecipitation, Intracellular Signaling Peptides and Proteins/chemistry, Leucine Zippers, Models, Genetic, Mutagenesis, Mutation, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins/chemistry, Recombinant Fusion Proteins/chemistry, Transfection
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