Leucine Zipper-mediated Homo-oligomerization Regulates the Rho-GEF Activity of AKAP-Lbc

• Published in: The Journal of biological chemistry Vol. 280; no. 15; pp. 15405 - 15412
• Main Authors: Baisamy, Laurent, Jurisch, Nathalie, Diviani, Dario
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2005; American Society for Biochemistry and Molecular Biology
• Subjects: 14-3-3 Proteins - chemistry; A Kinase Anchor Proteins; Adaptor Proteins, Signal Transducing - chemistry; Amino Acid Motifs; Apoptosis Regulatory Proteins; Blotting, Western; Cell Line; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; Glutathione Transferase - metabolism; Green Fluorescent Proteins - metabolism; GTP-Binding Protein alpha Subunits, G12-G13 - chemistry; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors - chemistry; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins - chemistry; Leucine Zippers; Minor Histocompatibility Antigens; Models, Genetic; Mutagenesis; Mutation; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins - chemistry; Recombinant Fusion Proteins - chemistry; Rho Guanine Nucleotide Exchange Factors; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M414440200

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 α-subunit of the heterotrimeric G protein G12, 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.