Abstract
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.
Highlights
Compartmentalization of signaling molecules through association with anchoring and scaffolding proteins is a mechanism that ensures specificity of transduction events involved in cellular regulation
The C-terminal Region of A-kinase anchoring proteins (AKAPs)-Lbc Negatively Regulates Basal Rho-guanine nucleotide exchange factor (GEF) Activity—We have demonstrated recently that AKAP-Lbc displays a low basal Rho-GEF activity in serumstarved cells, which can be significantly enhanced by the deletion of the N-terminal region of the anchoring protein upstream of the Dbl homology (DH) domain
Our previous results could not clearly determine whether the C-terminal region downstream of the pleckstrin homology (PH) domain regulates the function of AKAP-Lbc, because we found that the truncated form of AKAP-Lbc missing the N-terminal regulatory region displays a basal constitutive RhoGEF activity comparable with that of a deletion mutant of AKAP-Lbc missing both N and C termini [7]
Summary
Compartmentalization of signaling molecules through association with anchoring and scaffolding proteins is a mechanism that ensures specificity of transduction events involved in cellular regulation. Each AKAP contains a conserved amphipathic helix of 14 –18 residues that binds to the regulatory subunit dimers of the PKA holoenzyme [2,3,4] and displays a unique targeting motif that directs PKA1⁄7AKAP complexes to specific subcellular sites [5] Another fundamental role of AKAPs is to assemble signaling complexes by associating with multiple enzymes such as kinases, phosphatases, and other regulatory proteins. Many members of the Dbl family of GEF are maintained in a basal inactive conformation by intramolecular interactions involving the DH and PH domains as well as regulatory sequences Such interactions have been proposed to block the access of Rho GTPases to the DH domain and/or suppress the GEF activity of the exchange factor [13]. It appears that oligomerization can affect the functional properties of Dbl family members, the molecular mechanisms through which oligomerization regulates the activity of these exchange factors are poorly understood
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
