Regulating small G protein signaling to coordinate axon adhesion and repulsion

Abstract

Small GTPases play critical roles in diverse biological events including regulating both the cytoskeletal and adhesive properties of cells. The importance of small GTPases to these events stems from their ability to be turned on and off, respectively, by specific GEFs and GAPs. In neurons, for example, regulation of small GTPase activity by extracellular guidance cues controls axonal and dendritic process shape, extension and navigation. Here, we discuss recent findings that indicate a specific regulator of small GTPase signaling, the Plexin transmembrane GAP, is differentially controlled by specific extracellular cues to guide growing axons. In particular, Plexins are receptors for one of the largest families of axon guidance cues, Semaphorins and negatively regulate cell morphology and motility by serving as GAPs for Ras/Rap family GTPases. Recent observations reveal that Plexin's GAP activity is controlled by the cAMP-dependent protein kinase (PKA), which phosphorylates Plexin and generates a binding site for the phospho-serine/threonine binding protein 14-3-3ε. This PKA-mediated Plexin-14-3-3ε interaction prevents Plexin from associating with its GTPase substrate, and thus antagonizes Semaphorin signaling. We now further examine these interactions and how they provide a new logic by which axon guidance signaling pathways over-ride one another to steer growing axons. We also further explore how Plexin interacting proteins, including Ras, PKA and 14-3-3 may interact with the Plexin GAP domain. Our observations also further indicate that 14-3-3 proteins may have conserved roles in the regulation of GTPase activity.

Keywords: 14-3-3; Drosophila; GAP; Plexin receptor signaling; motor axon guidance; protein kinase A; semaphorin.

Figure 1. Working model of the mechanisms underlying Drosophila Sema-1a/PlexA-mediated repulsive axon guidance. (A) Drosophila Sema-1a exerts repulsive guidance effects by activating PlexA, which then induces repulsion. In particular, the cytoplasmic region of PlexA interacts with Mical, a novel F-actin disassembly factor that directly oxidizes the Met44 residue of actin to disassemble F-actin and limit actin polymerization. Plexin family proteins also have a GAP domain within their cytoplasmic region, which inactivates members of the Ras/Rap family of small GTPases to inhibit Integrin-mediated cell adhesion (De-Adhesion). Our recent results indicate that this Sema/Plexin “De-adhesion” can be turned-off by phosphorylation of the GAP domain of PlexA by protein kinase A (PKA), which allows binding of 14-3-3ε to PlexA and disrupts the interaction of the PlexA GAP domain with its Ras substrate. These interactions effectively allow Integrin-mediated adhesion to be turned back on (see also B). Several other molecules including Off-track, Gyc76C and Plexin B have also been implicated in this signaling pathway but their mechanistic role in this pathway is not yet clear. The circled numbers (1, 2 and 3) refer to the panels in (B) which provide more detail. (B) Panel 1: Plexin RasGAP facilitates GTPase activity of its substrate Ras/Rap family GTPases. Subsequently, GDP-bound inactive Ras is unable to activate Integrin-mediated cell adhesion. Panel 2: PKA phosphorylates a serine residue in the Plexin RasGAP domain and generates a binding site for 14-3-3ε. Panel 3: The interaction between 14-3-3ε and Plexin inhibits Plexin GAP activity by preventing it from associating with its Ras substrate. GTP-bound active Ras is able to activate Integrin-mediated adhesion.

Figure 2. Structural Comparison of Proteins Interacting with the Plexin GAP domain. Our results indicate that Ras family GTPases, 14-3-3ε, and a catalytic subunit of PKA (Pka-C) share a binding site within the GAP domain of Plexin A. The conserved arginine residues that are critical for Plexin RasGAP activity are labeled in red. The residue corresponding to a 14-3-3ε binding site is labeled in yellow, which is veiled by the arginine residues. The amino acid residues for these arginine and serine in both mouse Plexin A3 (m) and Drosophila PlexA (d) are indicated by arrows. The structural model of the Plexin A3 cytoplasmic region (some portions of the cytoplasmic region were not resolved in the crystal structure) is oriented with the concave surface of RasGAP domain facing downward. Active sites for proteins that interact with the Plexin RasGAP domain are indicated such as GTP for R-Ras and ATP for Pka-C. The phospho-serine-binding pocket of 14-3-3 is also indicated (14-3-3 is thought to often function as a dimer and bind two different phosphorylated residues. For clarity, a monomer of 14-3-3 is depicted). Protein data bank identification numbers: 3IG3 for mouse Plexin A3, 2FN4 for human R-Ras, 2F7X for cow catalytic subunit of PKA and 2BR9 for human 14-3-3ε.