Leucine zipper-mediated homo-oligomerization regulates the Rho-GEF activity of AKAP-Lbc.
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.
Baisamy
Laurent
L
Département de Pharmacologie et de Toxicologie, Faculté de Médecine, Lausanne 1005, Switzerland.
Jurisch
Nathalie
N
Diviani
Dario
D
D048948:14-3-3 Proteins / Q000737:chemistry; D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry; D020816:Amino Acid Motifs; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000737:chemistry; D019204:GTP-Binding Proteins; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D016350:Leucine Zippers; D015778:Minor Histocompatibility Antigens; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D064067:Rho Guanine Nucleotide Exchange Factors; D014162:Transfection
D048948:14-3-3 Proteins; D054758:A Kinase Anchor Proteins; C085467:AKAP13 protein, human; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D015778:Minor Histocompatibility Antigens; D011518:Proto-Oncogene Proteins; C100494:RTKN protein, human; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13