14-3-3ε Couples Protein Kinase A to Semaphorin Signaling and Silences Plexin RasGAP-Mediated Axonal Repulsion

Abstract

Summary The biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are among the largest families of axon guidance cues and utilize Plexin (Plex) receptors to exert repulsive effects on axon extension. However, Semaphorin repulsion can be turned-off by other distinct cues and signaling cascades, raising questions of the logic underlying these events. We now uncover a simple biochemical switch that controls Semaphorin/Plexin repulsive guidance. Plexins are Ras family GTPase activating proteins (GAPs) and we find that the PlexA GAP domain is phosphorylated by the cAMP-dependent protein kinase (PKA). This PlexA phosphorylation generates a specific binding site for 14-3-3ε, a phospho-binding protein that we find to be necessary for axon guidance. These PKA-mediated Plexin-14-3-3ε interactions prevent PlexA from interacting with its Ras family GTPase substrate and antagonize Semaphorin repulsion. Our results indicate that these interactions switch repulsion to adhesion and identify a point of convergence for multiple guidance molecules.

Genetic dissection of plexin signaling in vivo

“…Surprisingly, although plexins have been shown to regulate several independent signaling pathways in vitro, mutations in the GAP domain were sufficient to fully phenocopy the null mutants in vivo with respect to development of the nervous, the vascular, and the skeletal system. This crucial importance of the GAP domain of plexins during mouse development is in line with findings in Drosophila and Caenorhabditis elegans, where the GAP domains of PlexA and PLX-1 have been shown to be critical for development of the nervous system (47,48). Owing to the perinatal lethality of Plexin-B2 and Plexin-D1 GAP domain mutant mice, we could not systematically address the functional significance of the GAP domain at postnatal stages of development or in physiological and pathophysiological processes in the adult [e.g., for Plexin-B2 in the postnatal migration and proliferation of neuroblasts (26) and in wound healing (49), or for Plexin-D1 in postnatal development of the nervous system (34,35) and in angiogenesis (36,50)].…”

Regulating small G protein signaling to coordinate axon adhesion and repulsion

“…27,28 Interestingly, in contrast to Mical, multiple genetic interaction experiments indicate that Nervy and PKA antagonize Sema/PlexAmediated repulsive axon guidance. 27,29 These cAMP-dependent protein kinase (PKA)-mediated antagonistic effects on PlexA-dependent repulsion is reminiscent of observations revealing that increasing cAMP can silence the repulsive effects of specific Semas on cultured Xenopus and chick growth cones (Reviewed in 8,29 ). Thus, this conserved role of cAMP signaling raises interesting questions about the substrates of PKA and how PKA inactivates Sema/Plexin repulsive signaling.…”

“…29 Members of the 14-3-3 family of proteins are well-known for their interactions with phosphorylated serine/ threonine residues in target proteins and play important roles in cellular signaling. 30,31 Interestingly, our analysis of the interaction between PlexA and 14-3-3ε revealed that a single serine residue situated within the Plexin RasGAP domain was critical for the PlexA-14-3-3ε interaction.…”

“…30,31 Interestingly, our analysis of the interaction between PlexA and 14-3-3ε revealed that a single serine residue situated within the Plexin RasGAP domain was critical for the PlexA-14-3-3ε interaction. 29 Thus, we wondered if this interaction between PlexA and 14-3-3ε could regulate Plexin RasGAP-mediated axon guidance.…”

“…29) but their in vivo roles in axon guidance have only been poorly defined. Using the Drosophila nervous system as a model, we found that 14-3-3ε is highly expressed within developing neurons and their axonal processes and can be co-immunoprecipitated with neuronal PlexA in vivo.…”