Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neuron. Author manuscript; available in PMC 2013 December 9.
Published in final edited form as:
PMCID: PMC3857333

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


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.


Neural connections form during development when neurons extend stalk-like axonal appendages that actively explore their environment, seeking specific signals that will guide them to their targets. Work over the past twenty years has identified a number of these extracellular signals, revealing that specific attractive and repulsive guidance cues control the cytoskeletal and adhesive machinery necessary for axon elongation (Kolodkin and Tessier-Lavigne, 2011). More recently, transmembrane receptors and intracellular signaling molecules have been found for many of these guidance cues, providing a further understanding of the molecular biology of axon guidance (Kolodkin and Tessier-Lavigne, 2011; Bashaw and Klein, 2010). Yet, these fundamental discoveries have also raised important new questions regarding the biochemical mechanisms that enable growing axons to choose among this diverse array of guidance information, much of which is presented in concert, to precisely navigate to their targets.

Semaphorins (Semas) are among the largest families of axon guidance cues and are best known for their ability to sculpt the nervous system by serving as axonal repellents (Kolodkin and Tessier-Lavigne, 2011). Semas exert their repulsive effects by disassembling the actin and microtubule cytoskeletal elements necessary for axonal extension, as well as by disrupting the adhesive interactions between an axon and its substrate (Hung and Terman, 2011). Semas utilize Plexin receptors to exert their cell biological effects and recently a number of signaling molecules have been identified that mediate Sema/Plexin effects on the cytoskeleton (Zhou et al., 2008; Bashaw and Klein, 2010), including a novel actin disassembly factor, Mical (Hung et al., 2011; Hung et al., 2010). Interestingly, Plexins also directly associate with small GTP-binding proteins and contain a GTPase activating protein (GAP) domain within their cytoplasmic portions (Rohm et al., 2000; Vikis et al., 2000; Driessens et al., 2001; Hu et al., 2001; Oinuma et al., 2004; He et al., 2009; Tong et al., 2009). These observations have provided a direct link between Semas/Plexins and small GTP-binding proteins, which are key regulators of cytoskeletal dynamics and cell adhesion (Hall and Lalli, 2010). Indeed, in vitro work has indicated that Plexins exert repulsive/de-adhesive effects on growing axons by employing their RasGAP activity to inhibit Ras/Integrin-dependent axon-substrate adhesion (Oinuma et al., 2004; Toyofuku et al., 2005; Oinuma et al., 2006; Uesugi et al., 2009; Tong et al., 2009).

Growing evidence also indicates that the repulsive effects of axon guidance cues can be silenced and even turned into attraction by raising the levels of specific signaling molecules like cyclic nucleotides. cAMP, for example, has emerged as a potent antirepellent that enables axonal growth and regeneration on repulsive/inhibitory substrates including Semas (Song et al., 1998; Cai et al., 1999; Hopker et al., 1999; Dontchev and Letourneau, 2002; Neumann et al., 2002; Qiu et al., 2002; Chalasani et al., 2003; Pearse et al., 2004; Han et al., 2007; Xu et al., 2010). The molecular and biochemical mechanisms of this cAMP antirepellent action are still poorly understood, but it is interesting that the cAMP-dependent protein kinase (PKA), which is activated by cAMP, has been found to associate in a complex with the Sema receptor Plexin (Terman and Kolodkin, 2004; Fiedler et al., 2010) and antagonize Sema-mediated repulsive axon guidance (Dontchev and Letourneau, 2002; Chalasani et al., 2003; Terman and Kolodkin, 2004; Parra and Zou, 2010). The targets of PKA and its biochemical role in regulating Sema/Plexin repulsive axon guidance are unknown.

We now find that PKA phosphorylates a specific serine residue within the Plexin RasGAP domain and generates a binding site for a member of the 14-3-3 family of phosphoserine binding proteins, 14-3-3ε. Moreover, these PKA-mediated 14-3-3ε–Plexin interactions occlude the association between Plexin and its RasGAP substrate, Ras2, concomitantly making axons less responsive to Sema-mediated repulsion and more responsive to Integrin-mediated adhesion. Our findings, therefore, uncover both a new molecular integration point between important axon guidance signaling pathways and a biochemical logic by which this guidance information is coalesced to steer the growing axon.


14-3-3ε is Neuronal PlexA–interacting Protein

The C-terminal region of the 14-3-3ε protein was identified as a strong Drosophila Plexin A (PlexA) interactor in a yeast two-hybrid interaction screen (Figures 1Aa-c). 14-3-3 protein family members are important regulators of signal transduction through their ability to bind to phosphorylated serine/threonine residues within target proteins (Figure 1Ab; (Tzivion et al., 2001; Yaffe and Elia, 2001)). Drosophila contains two highly conserved 14-3-3 family members (also called Par-5 proteins), 14-3-3ε and 14-3-3ζ/leonardo (Figure S1A), but PlexA selectively interacted with only 14-3-3ε in our yeast interaction assay (Figure S1B). Likewise, we saw selective interactions between neuronally-expressed HAPlexA and purified recombinant GST-14-3-3ε protein (Figure 1Ad). The other Drosophila Plexin, PlexB, did not interact with 14-3-3ε in our yeast interaction assay (Figure 1Ac), also suggesting a specificity among PlexA–14-3-3ε interactions. Further analyses revealed that 14-3-3ε, like PlexA, was highly expressed in the embryonic brain and nerve cord (Figures 1Ba-c, S1C) and localized strongly to central nervous system (CNS) and motor axons (Figures 1Bc’-c”). 14-3-3ε was also consistently detected in the complex immunoprecipitated by neuronal HAPlexA but not by non-specific controls (Figure 1Bd). These results, in conjunction with other related binding experiments (Figures 5D, ,8B,8B, S4B, S7E), indicate that PlexA and 14-3-3ε form a complex in neurons.

Figure 1
14-3-3ε is a Neuronal PlexA Interacting Protein
Figure 5
PlexA–14-3-3ε Interactions Occur Through a Single PlexA Phosphoserine Residue
Figure 8
PlexA–14-3-3ε Interactions Block Plexin Ras-GAP-mediated Repulsive Axon Guidance

14-3-3ε is Required for Axon Pathfinding

To begin to explore the function of the PlexA–14-3-3ε physical interaction, we turned to the Drosophila embryonic nervous system. A number of 14-3-3ε loss-of-function (LOF) alleles have been well-characterized (Figure S2A; (Chang and Rubin, 1997; Acevedo et al., 2007)) and have revealed that 14-3-3ε mutants do not exhibit overt morphological defects within the nervous system or musculature (Acevedo et al., 2007). Maternally supplied 14-3-3ε and compensation by 14-3-3ζ are sufficient for many developmental processes including cell fate specification and patterning (Chang and Rubin, 1997; Su et al., 2001; Acevedo et al., 2007; Krahn et al., 2009). However, neuronal expression of 14-3-3ε is necessary for normal embryonic hatching and adult viability for unknown reasons (Acevedo et al., 2007). Therefore, we wondered if 14-3-3ε LOF mutants exhibited axon guidance defects, and employed well-characterized Drosophila CNS and motor axons to test this possibility. For instance, axons within the Drosophila Intersegmental Nerve b (ISNb) motor axon pathway normally defasciculate from the pioneering ISN to innervate their muscle targets including muscles 6/7 and 12/13 (Figures 2A-B). In contrast, we found that ISNb axons within multiple combinations of 14-3-3ε LOF mutants exhibited specific and highly penetrant axon guidance defects including abnormal defasciculation, inappropriate pathway selection, and decreased muscle innervation (Figures 2C-E, S2B and E). These ISNb pathfinding defects were significantly rescued upon restoration of 14-3-3ε expression in 14-3-3ε mutants using a FLAG14-3-3ε transgene (Figures 2A, E, S2D). We also observed axonal pathfinding errors within other motor axon pathways of 14-3-3ε LOF mutants, including the Segmental Nerve A (SNa) (Figures 2D-E, S2B), as well as in the CNS (Figure S2C). These results reveal that a member of the 14-3-3 family of phosphoserine binding proteins, 14-3-3ε, is required for axon guidance in vivo.

Figure 2
14-3-3ε is Required for Axon Guidance

14-3-3ε Antagonizes Sema/Plex-mediated Repulsive Axon Guidance

We next compared 14-3-3ε–dependent axon guidance defects to those resulting from manipulating Sema-1a/PlexA signaling. LOF alleles of PlexA, its ligand Sema1a, and its signaling component Mical, generate motor axon pathfinding defects characterized by increased axonal fasciculation, stalling, and abnormal muscle innervation (Yu et al., 1998; Winberg et al., 1998b; Terman et al., 2002; Hung et al., 2010). Interestingly, while some of the axon guidance defects we observed in 14-3-3ε mutants were similar to Sema1a, PlexA, and Mical mutants (Figure 2F), a majority were characterized by increased axonal defasciculation and resembled the effects of increasing Sema/PlexA/Mical repulsive axon guidance (Figures 2F, S2E). Furthermore, neuronal overexpression of 14-3-3ε generated axon guidance defects that resembled decreasing Sema/PlexA/Mical repulsive axon guidance (Figures 2F, S2B). These observations suggest that similar to Sema-1a/PlexA signaling components such as Nervy and the regulatory subunit of PKA (PKA RII) (Figure 2F; (Terman and Kolodkin, 2004)), 14-3-3ε may antagonize Sema-1a/PlexA repulsive axon guidance.

To further address this possibility, we turned to enhancer-supressor genetic assays dependent on Sema-1a/PlexA repulsive axon guidance. Ectopic expression of Sema-1a in muscles leads to reduced muscle innervation (Yu et al., 1998). These effects are due to the repulsive action of Sema-1a (Yu et al., 1998) and are suppressed by decreasing the levels of the Sema-1a receptor, PlexA (Winberg et al., 1998b). In contrast, decreasing the levels of 14-3-3ε enhanced Sema-1a repulsion (Figure 3B-D); suggesting that similar to PKA RII and Nervy (Figure 3C-D; (Terman and Kolodkin, 2004)), 14-3-3ε opposes Sema-1a repulsion. To further investigate these antagonistic interactions, we turned to genetic assays dependent on the repulsive effects of PlexA. Increasing the levels of neuronal PlexA generates abnormally defasciculated axons that result in discontinuous CNS longitudinal connectives and axons crossing the midline or projecting abnormally into the periphery (Figures 4A, S3A; (Winberg et al., 1998b; Ayoob et al., 2004)). Strikingly, decreasing the levels of 14-3-3ε significantly increased these PlexA-dependent guidance defects (Figure 4A-C), while increasing neuronal 14-3-3ε significantly decreased these PlexA-dependent guidance defects (Figure 4B-C). Together, these results along with other in vivo Sema1a/PlexA-dependent CNS and motor axon guidance assays (Figure S3), indicate that 14-3-3ε antagonizes Sema1a/PlexA-mediated repulsive axon guidance.

Figure 3
14-3-3ε/PKA/Integrin Signaling Antagonizes Sema-1a Repulsive Axon Guidance
Figure 4
14-3-3ε Antagonizes PlexA Repulsive Axon Guidance

A Single Phosphorylated Serine Residue on PlexA Mediates Its Interaction with 14-3-3ε

To begin to investigate the mechanism by which 14-3-3ε antagonizes Sema-1a/PlexA-mediated repulsive axon guidance, we sought to determine the site of interaction between PlexA and 14-3-3ε. We found that the portion of PlexA that was necessary and sufficient for the interaction with 14-3-3ε contained a consensus 14-3-3 binding sequence (Figure 5A-B). In particular, 14-3-3 proteins typically bind to single phosphorylated serine or threonine residues on target proteins (Yaffe and Elia, 2001) and Drosophila PlexA contains a mode I 14-3-3 consensus binding motif, R/KSXpSXP, where p represents the phosphorylated serine (Ser1794) residue predicted to mediate the interaction with 14-3-3 proteins (Figure 5B; (Yaffe et al., 1997; Rittinger et al., 1999)). To test this possibility, we substituted alanine (Ala) for serine (Ser) and threonine (Thr) residues within this consensus 14-3-3ε binding motif. We found that the predicted Ser1794 residue was necessary for the observed PlexA interaction with 14-3-3ε (Figures 5C, S4A). Next, we generated phospho-mimetic forms of Ser1794 (Ser1794 to Glu1794 or Asp1794), but found that as with other 14-3-3 interacting proteins adding one negative charge was not sufficient for the interaction between PlexA and 14-3-3ε (Figures 5C, S4A). Therefore, we turned to in vitro binding assays with PlexA and purified 14-3-3ε to see if phosphorylation was critical for the PlexA–14-3-3ε interaction. Indeed, we found that phophatase treatment disrupted the ability of neuronal HAPlexA to associate with 14-3-3ε (Figures 5D, S4B). Taken together, these results indicate that PlexA and 14-3-3ε associate via a single phosphoryated serine residue present in the cytoplasmic portion of the PlexA receptor (Figure 5E).

PKA Phosphorylates PlexASer1794 and Enables the PlexA–14-3-3ε Interaction

So what might be the kinase that phosphorylates PlexA at Ser1794? Interestingly, PlexA and 14-3-3ε interact in yeast indicating that a serine/threonine kinase present in yeast is sufficient to phosphorylate PlexA. We also noticed that PlexA’s 14-3-3ε binding site contained a consensus phosphorylation site (R/KxxS; Figures 6A, S5A) for several kinases well-conserved from yeast to humans including PKA, the Ca2+-dependent protein kinase (PKC), and the cGMP-dependent protein kinase (PKG). Therefore, we conducted in vitro kinase assays with purified proteins and found that PlexA (PlexACyto2) is specifically phosphorylated by two kinases, PKA and Cdk5 (Figures 6A, S5A-B). Mutating the PlexASer1794 residue significantly decreased this PKA-, but not Cdk5-, dependent phosphorylation (Figures 6B, S5C), revealing that the PlexA Ser1794 residue that is critical for 14-3-3ε binding is selectively phosphorylated by PKA. Likewise, our results indicated that PKA is sufficient to mediate this PlexASer1794–14-3-3ε interaction, since activating PKA signaling with forskolin significantly enhanced the association between FLAG14-3-3ε and HAPlexA in a Ser1794-dependent manner (Figure 6C). We thus wondered if PKA was necessary for phosphorylating PlexASer1794 in vivo. Employing a rabbit polyclonal antibody that we generated that selectively recognized the phosphorylated form of PlexASer1794 (phospho-PlexAS1794) (Figures 6D-E, S5D-E), we found that decreasing the levels of PKA in vivo significantly reduced the levels of phospho-PlexAS1794 (Figure 6F). Therefore, our results indicate that PlexASer1794 is phosphorylated by PKA, which mediates the interaction between PlexA and 14-3-3ε (Figure 6H).

Figure 6
PKA Phosphorylates PlexASer1794 Mediating the PlexA–14-3-3ε Interaction

PKA-mediated 14-3-3ε Interactions with PlexASer1794 Antagonize Sema/Plex-dependent Axon Repulsion

A protein complex containing PKA has previously been found to associate with the PlexA receptor (Terman and Kolodkin, 2004; Fiedler et al., 2010). This work in combination with our new biochemical results suggest a model in which inactive PKA is tethered to the PlexA receptor and upon cAMP-mediated activation, PKA phosphorylates PlexA at Ser1794 and provides a binding site for 14-3-3ε. We therefore wondered what is the role of this PKA–14-3-3ε interaction in Sema-1a/PlexA repulsive axon guidance. Similar to loss of 14-3-3ε, decreasing PKA catalytic activity increased Sema-1a/PlexA repulsive axon guidance (Figures 3C-D, ,6G,6G, S3A-B). These effects were further enhanced by simultaneously decreasing PKA and 14-3-3ε (Figures 6G, S3B), indicating that PKA and 14-3-3ε work together to antagonize PlexA repulsive axon guidance. These results also predict that disrupting the interaction between 14-3-3ε and PlexA might generate a hyperactive PlexA receptor so we generated two transgenic fly lines (HAPlexAS1794A (SA) and HAPlexAS1794E (SE); Figure 7A) containing single mutations that disrupted the assocation between PlexA and 14-3-3ε. Notably, although both the SA and SE PlexA receptors were expressed at or below the levels of HAPlexA (WT) in neurons and on axonal surfaces (Figures 7B, S6A-B; see also Figure S7A), both mutations produced guidance defects consistent with increased PlexA repulsion (Figure 6C-D). Therefore, disrupting the interaction between PlexA and 14-3-3ε generates hyperactive Sema-1a/PlexA-mediated repulsive axon guidance signaling.

Figure 7
Disrupting the PlexA–14-3-3ε Interaction Generates Hyperactive PlexA Repulsive Axon Guidance

PlexA–14-3-3ε Interactions Block Plexin RasGAP-Mediated Repulsive Axon Guidance

The Ser1794 residue that is critical for the interaction between 14-3-3ε and PlexA is located adjacent to one of the enzymaticaly critical arginine residues (Arg1798) through which Plexins turn-off RasGTP signaling (Figure 8A). In particular, the intracellular region of Plexins contains a GAP enzymatic domain that is structurally and functionally similar to GAPs for Ras superfamily GTPases (Figure 8A; (Oinuma et al., 2004; He et al., 2009; Tong et al., 2009; Bell et al., 2011)). As a RasGAP, Plexin facilitates endogenous GTP hydrolysis by Ras family GTPases and thus functions to antagonize or turn-off RasGTP signaling. In Plexins, like other RasGAPs, arginine fingers cooperatively confer both GTPase binding and GAP activity, suggesting that the association of 14-3-3ε with PlexASer1794 would likely perturb the association between PlexA and its substrate GTPase (Figure 8A; (He et al., 2009; Tong et al., 2009)). To begin to test this mechanism of action, we made point mutations disrupting the catalytically important arginine fingers of PlexA (HAPlexARA (RA); Figure 7A). Neuronal expression of the PlexA GAP-deficient protein failed to rescue PlexA-/- mutant axon guidance defects (Figure S6C) and suppressed the ability of PlexA to mediate repulsive axon guidance (Figures 7C-D). Thus, as has been previously described in vitro (Rohm et al., 2000; Oinuma et al., 2004; He et al., 2009), the GAP activity of PlexA is also important in vivo for repulsive axon guidance.

Plexin family members utilize Ras family GTPases including R-Ras and M-Ras as substrates (Oinuma et al., 2004; Toyofuku et al., 2005; Saito et al., 2009), and we found that Drosophila PlexA also preferentially associated with the GTP bound form of the Drosophila R-Ras orthologue, Ras2 (Figures 8B, S7B-C), and facilitated GTP hydrolysis (Figure S7D). Likewise, using in vivo genetic assays, we found that constituitively active Ras2, but not Ras1, suppressed PlexA-mediated repulsive axon guidance (Figure S3A), further indicating that Ras2 specifically plays a role in PlexA repulsive signaling. Therefore, since both 14-3-3ε and Ras2 associate with the same region of the PlexA receptor, the RasGAP domain (Figure 8A), we wondered if 14-3-3ε disrupted the PlexA–Ras2 association. Using biochemical approaches, we found that the interactions between HAPlexA and Ras2GTP were selectively decreased in the presence of purified 14-3-3ε (Figure 8B), as was the GAP activity of PlexA towards Ras2GTP (Figure S7E-F). Moreover, mutating the 14-3-3ε-binding site of PlexA prevented 14-3-3ε from altering these PlexA-Ras associations (Figure S7G-H), further indicating that 14-3-3ε blocks the association between PlexA and Ras2. Therefore, we wondered if the increased PlexA-dependent repulsive axon guidance defects that we observed in 14-3-3ε LOF mutants (Figure 4) might result from increased PlexA GAP-Ras2 interactions, and thus a decrease in the amount of active Ras2 in the vicinity of the PlexA receptor. Indeed, we found that raising the levels of active Ras2 in neurons suppressed the hyperactive PlexA repulsion caused by loss of 14-3-3ε (Figure 8C), indicating that PlexA–14-3-3ε interactions function to silence PlexA RasGAP-mediated repulsive axon guidance.

A 14-3-3ε-dependent Switch Regulates Sema/Plex-Mediated Repulsion and Ras/Integrin-Mediated Adhesion

R-Ras signaling has been found to induce an increase in cellular adhesion and migration by activating integrins to bind to their ECM ligands (Zhang et al., 1996; Keely et al., 1999; Ivins et al., 2000; Oinuma et al., 2006). Plexins exert their repulsive/de-adhesive effects on growing axons by employing their GAP activity to inhibit specific Ras family GTPases and thereby turn-off Integrin-mediated substrate adhesion (Oinuma et al., 2004; Oinuma et al., 2006). Thus, our results suggest that this Ras/Integrin-dependent adhesion can be turned-back-on through PKA-mediated phosphorylation of the PlexA RasGAP domain and subsequent binding of 14-3-3ε. Consistent with such a mechanism of action, integrins play important roles in establishing normal axonal trajectories and the loss of integrins generates axon guidance defects that resemble those seen following manipulations of Sema-1a/PlexA signaling (Hoang and Chiba, 1998; Huang et al., 2007; Stevens and Jacobs, 2002). Furthermore, we found that increasing the neuronal levels of integrins suppressed Sema-1a/PlexA repulsive axon guidance (Figure S3A), while decreasing integrin levels enhanced Sema-1a/PlexA repulsive effects (Figures 3C-D, S3A). Moreover, increasing specific integrins (α2 Integrin (αPS2); (Stevens and Jacobs, 2002)) in neurons suppressed the hyperactive PlexA repulsive signaling caused by the loss of 14-3-3ε (Figure 8C). Therefore, we reasoned that if 14-3-3ε functions to increase Ras/Integrin-mediated adhesion then the axon guidance defects we observe in 14-3-3ε mutants might be significantly rescued by increasing Ras/Integrin signaling. Indeed, we found that the ISNb and SNa motor axon pathfinding defects present in 14-3-3ε mutants were rescued by expressing constituitively active Ras2 (Figure 9A) or specific integrins (Figure 9B). Together, these results reveal that raising the levels of Ras/Integrin signaling counteracts the effects of decreasing 14-3-3ε, indicating a cAMP/PKA/14-3-3ε signaling pathway that directly controls the balance between Plexin RasGAP-mediated repulsion and Ras/Integrin-mediated adhesion.

Figure 9
Increasing Ras2–Integrin Adhesive Signaling Rescues 14-3-3ε Mutant Axon Guidance Defects


Axons rely on the activation of guidance receptors for correct navigation but receptor inactivation is also thought to be a means through which growth cones integrate both attractive and repulsive guidance signals. Our results indicate that such a mechanism plays a critical role in Sema/Plex-mediated repulsive axon guidance. We find that PlexA uses its RasGAP activity to specify axon guidance but this activity is antagonized by a PKA–mediated signaling pathway. PKA directly phosphorylates the GAP domain of PlexA and this phosphorylation provides a binding site for 14-3-3ε. 14-3-3ε is critical for axon guidance and disrupts the ability of PlexA to interact with its Ras GTPase substrate. These interactions effectively switch PlexA-mediated axonal repulsion to Integrin-mediated adhesion and provide a simple biochemical mechanism to integrate antagonistic axon guidance signals (Figure 10).

Figure 10
Silencing of Plexin RasGAP-mediated Axon Repulsion by PKA/14-3-3ε

Our genetic experiments identify a critical role for 14-3-3ε proteins in directing axon guidance events during development. The 14-3-3 proteins are a phylogentically well-conserved family of cytosolic signaling proteins including seven mammalian members that play key roles in a number of cellular processes (Tzivion et al., 2001; Yaffe and Elia, 2001). Interestingly, 14-3-3 family proteins were first identified because of their high level of expression in the brain (Aitken, 2006), but despite considerable interest in their functions (Skoulakis and Davis, 1998; Berg et al., 2003), their roles in the nervous system are still incompletely understood. For instance, 14-3-3 proteins are highly expressed in growing axons and have been found to modulate neurite extension and growth cone turning in vitro in a number of contexts (Nozumi et al., 2009; Yoon et al., 2011; Kent et al., 2010). However, their necessity for directing axonal growth and guidance events in vivo are unknown as is the functional role of each family member in these neurodevelopmental processes. We now find that one of the two Drosophila 14-3-3 family members, 14-3-3ε, is required in vivo for axon guidance and plays specific roles in the pathfinding of motor and CNS axons. Moreover, previous mutant analysis has revealed that the other 14-3-3 family member in Drosophila, 14-3-3ζ (Leonardo), does not exhibit significant motor axon guidance or innervation defects (Broadie et al., 1997) but plays a critical role in synaptic transmission and learning and memory (Skoulakis and Davis, 1996; Broadie et al., 1997). These results indicate that individual 14-3-3 family members play specific roles in the development of the nervous system and in light of the requirement of 14-3-3ε in mammalian brain development and neuronal migration (Toyo-oka et al., 2003), and potential roles for 14-3-3ε (YWHAE) in human neurological disease (Mignon-Ravix et al., 2010; Bi et al., 2009; Nagamani et al., 2009; Schiff et al., 2010), future work will determine if 14-3-3ε’s role in axon guidance is phylogenetically conserved.

Our genetic and biochemical experiments also identify a specific role for 14-3-3ε in regulating Sema/Plex-mediated repulsive axon guidance. Sema/Plex-mediated repulsive axon guidance is antagonized by increasing cAMP levels (Song et al., 1998; Dontchev and Letourneau, 2002; Chalasani et al., 2003; Parra and Zou, 2010), but the mechanisms underlying these cAMP-mediated effects are poorly understood. Interestingly, Plexins associate with the cAMP-dependent protein kinase (PKA) via MTG/Nervy family PKA (A kinase) anchoring proteins (AKAPs) (Fukuyama et al., 2001; Schillace et al., 2002; Terman and Kolodkin, 2004; Fiedler et al., 2010; Corpora et al., 2010). AKAPs position PKA at defined locations to allow for the spatially and temporally specific phosphorylation of target proteins in response to local increases in cAMP (Wong and Scott, 2004) and we now find that PKA phosphorylates the cytoplasmic portion of PlexA. Our genetic and biochemical results suggest that this phosphorylation provides a binding site for a specific 14-3-3 family member, 14-3-3ε. 14-3-3 proteins are well-known as phosphoserine/threonine-binding proteins and have been found to utilize this ability to regulate the activity of specific enzymes (Yaffe and Elia, 2001; Tzivion et al., 2001). We find that mutating the 14-3-3ε binding site on PlexA generates a hyperactive PlexA receptor, providing a better understanding of the molecular and biochemical events through which cAMP signaling regulates Sema/Plex repulsive axon guidance. Future work will focus on identifying the upstream extracellular signal that increases cAMP levels, although it is interesting that the axonal attractant Netrin is known to increase cAMP levels (Corset et al., 2000; Nicol et al., 2011) and antagonize Sema-mediated axonal repulsion (Winberg et al., 1998a).

Our results also indicate that the GAP activity of PlexA is critical in vivo for repulsive axon guidance and that cAMP/PKA/14-3-3ε signaling regulates this Plexin RasGAP-mediated repulsion. Plexins are GAPs for Ras superfamily proteins and in vitro work has revealed that the RasGAP activity of Plexin is important for its signaling role (Oinuma et al., 2004; Oinuma et al., 2006; Ito et al., 2006; Saito et al., 2009; Oinuma et al., 2010). We now find that RasGAP activity is required in vivo in neurons for Plex-mediated repulsive axon guidance. Moreover, our results indicate that 14-3-3ε binds to a single phosphoserine residue within the PlexA RasGAP domain and antagonizes PlexA RasGAP–mediated axon guidance. Interestingly, positive regulation of GTPase signaling may be a conserved function for 14-3-3ε since it also increases the efficiency of Ras signaling during Drosophila eye development (Chang and Rubin, 1997) and 14-3-3 turns-off the activity of other known GAPs and enhances Ras signaling (e.g., (Feng et al., 2004; Benzing et al., 2000)). Therefore, our results suggest a model (Figure 10) in which Sema/Plex interactions activate PlexA RasGAP activity, which inactivates Ras and disables Integrin-mediated adhesion. However, these Sema/Plex-mediated effects are subject to regulation, such that increasing cAMP levels activates PlexA-bound PKA to phosphorylate PlexA and provide a binding site for 14-3-3ε. These PlexA–14-3-3ε interactions occlude PlexA RasGAP-mediated inactivation of Ras and restore Integrin-dependent adhesion.

In conclusion, we have identified a simple mechanism that allows multiple axon guidance signals to be incorporated during axon guidance. Neuronal growth cones encounter both attractive and repulsive guidance cues but the molecular pathways and biochemical mechanisms that integrate these antagonistic cues and enable a discrete steering event are incompletely understood. One way in which to integrate these disparate signals is to allow different axon guidance receptors to directly modulate each other’s function (e.g., (Stein and Tessier-Lavigne, 2001)). Another means is to tightly regulate the cell surface expression of specific receptors and thereby actively prevent axons from seeing certain guidance cues (e.g., (Kidd et al., 1998; Brittis et al., 2002; Keleman et al., 2002; Nawabi et al., 2010; Chen et al., 2008; Yang et al., 2009)). Still further results are not simply explained by relatively slow modulatory mechanisms like receptor trafficking, endocytosis, and local protein synthesis but indicate that interpreting a particular guidance cue is susceptible to rapid intracellular modulation by other, distinct, signaling pathways (e.g., (Song et al., 1998; Dontchev and Letourneau, 2002; Terman and Kolodkin, 2004; Parra and Zou, 2010; Xu et al., 2010)). Our results now indicate a means to allow for such intracellular signaling cross-talk events and present a new logic by which axon guidance signaling pathways over-ride one another. Given this molecular link between such key regulators of axon pathfinding as cyclic nucleotides, phosphorylation, and GTPases, our observations on silencing Sema/Plex-mediated repulsive axon guidance also suggest new approaches to neutralize axonal growth inhibition and encourage axon regeneration.

Experimental Procedures

Yeast Two-Hybrid Screening and Assay

Yeast two-hybrid set-up, protein expression analyses, and screening were performed following standard procedures (Terman et al., 2002).

Genetics, Phenotypes, Imaging, and Histochemistry

Drosophila husbandry, genetics, imaging, and characterization of axon guidance were performed using standard methods (Terman et al., 2002; Hung et al., 2010).

In Vitro Binding Assays and Co-immunoprecipitation

GST pull-down (Oinuma et al., 2004) and co-immunoprecipitation (Terman et al., 2002) assays were performed using standard approaches. GDP and GTPγS-preloading was assessed by GST pull-down assays using GST-RBD proteins (Diekmann and Hall, 1995; Benard et al., 1999).

Protein Purification

Standard approaches were used to purify proteins (Self and Hall, 1995; Hung et al., 2010).

In Vitro Kinase Assays

In vitro kinase assays were perfomed using [γ-32P] ATP (PerkinElmer) as a phosphate donor following the manufacturer’s recommendation for each kinase and as described (Bibb et al., 1999).

Phosphorylation-Specific PlexA Antibody Generation

A polyclonal antibody which specifically recognizes phosphorylated PlexAS1794 was generated using synthetic phosphopeptides and injecting into rabbits (Covance) using standard approaches (Flavell et al., 2006).

Supplementary Material



We thank J. Bibb, C. Cowan, E. Ross, and X. Zhang for their input and for generous use of their reagents and equipment. We also greatly appreciate members of the Terman lab, M. Benson, D. Brower, J. Brugarolas, M. Cobb, H. He, P. Hiesinger, D. Jun, E. Kim, A. Kolodkin, H. Krämer, G. Rubin, A. Spradling, D. St Johnston, W. Williamson, Berkeley Drosophila Genome Project, Bloomington and Japanese Stock Centers, Drosophila Genomics Resource Center, Development Studies Hybridoma Bank, FlyTrap, FlyBase, UT Southwestern peptide synthesis core facility, and the Korea Research Foundation (KRF-2005-C00113) for reagents and assistance. Support was provided by the NIH (MH085923) to J.R.T.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Acevedo SF, Tsigkari KK, Grammenoudi S, Skoulakis EM. In vivo functional specificity and homeostasis of Drosophila 14-3-3 proteins. Genetics. 2007;177:239–253. [PubMed]
  • Aitken A. 14-3-3 proteins: a historic overview. Semin Cancer Biol. 2006;16:162–172. [PubMed]
  • Ayoob JC, Yu HH, Terman JR, Kolodkin AL. The Drosophila receptor guanylyl cyclase Gyc76C is required for semaphorin-1a-plexin A-mediated axonal repulsion. J Neurosci. 2004;24:6639–6649. [PubMed]
  • Bashaw GJ, Klein R. Signaling from axon guidance receptors. Cold Spring Harb Perspect Biol. 2010;2:a001941. [PMC free article] [PubMed]
  • Bell CH, Aricescu AR, Jones EY, Siebold C. A Dual Binding Mode for RhoGTPases in Plexin Signalling. PLoS Biol. 2011;9:e1001134. [PMC free article] [PubMed]
  • Benard V, Bohl BP, Bokoch GM. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem. 1999;274:13198–13204. [PubMed]
  • Benzing T, Yaffe MB, Arnould T, Sellin L, Schermer B, Schilling B, Schreiber R, Kunzelmann K, Leparc GG, Kim E, et al. 14-3-3 interacts with regulator of G protein signaling proteins and modulates their activity. J Biol Chem. 2000;275:28167–28172. [PubMed]
  • Berg D, Holzmann C, Riess O. 14-3-3 proteins in the nervous system. Nat Rev Neurosci. 2003;4:752–762. [PubMed]
  • Bi W, Sapir T, Shchelochkov OA, Zhang F, Withers MA, Hunter JV, Levy T, Shinder V, Peiffer DA, Gunderson KL, et al. Increased LIS1 expression affects human and mouse brain development. Nat Genet. 2009;41:168–177. [PMC free article] [PubMed]
  • Bibb JA, Snyder GL, Nishi A, Yan Z, Meijer L, Fienberg AA, Tsai LH, Kwon YT, Girault JA, Czernik AJ, et al. Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons. Nature. 1999;402:669–671. [PubMed]
  • Brittis PA, Lu Q, Flanagan JG. Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target. Cell. 2002;110:223–235. [PubMed]
  • Broadie K, Rushton E, Skoulakis EM, Davis RL. Leonardo, a Drosophila 14-3-3 protein involved in learning, regulates presynaptic function. Neuron. 1997;19:391–402. [PubMed]
  • Cai D, Shen Y, De Bellard M, Tang S, Filbin MT. Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron. 1999;22:89–101. [PubMed]
  • Chalasani SH, Sabelko KA, Sunshine MJ, Littman DR, Raper JA. A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon pathfinding. J Neurosci. 2003;23:1360–1371. [PubMed]
  • Chang HC, Rubin GM. 14-3-3 epsilon positively regulates Ras-mediated signaling in Drosophila. Genes Dev. 1997;11:1132–1139. [PubMed]
  • Chen Z, Gore BB, Long H, Ma L, Tessier-Lavigne M. Alternative splicing of the Robo3 axon guidance receptor governs the midline switch from attraction to repulsion. Neuron. 2008;58:325–332. [PubMed]
  • Corpora T, Roudaia L, Oo ZM, Chen W, Manuylova E, Cai X, Chen MJ, Cierpicki T, Speck NA, Bushweller JH. Structure of the AML1-ETO NHR3-PKA(RIIalpha) complex and its contribution to AML1-ETO activity. J Mol Biol. 2010;402:560–577. [PMC free article] [PubMed]
  • Corset V, Nguyen-Ba-Charvet KT, Forcet C, Moyse E, Chedotal A, Mehlen P. Netrin-1-mediated axon outgrowth and cAMP production requires interaction with adenosine A2b receptor. Nature. 2000;407:747–750. [PubMed]
  • Diekmann D, Hall A. In vitro binding assay for interactions of Rho and Rac with GTPase-activating proteins and effectors. Methods Enzymol. 1995;256:207–215. [PubMed]
  • Dontchev VD, Letourneau PC. Nerve growth factor and semaphorin 3A signaling pathways interact in regulating sensory neuronal growth cone motility. J Neurosci. 2002;22:6659–6669. [PubMed]
  • Driessens MH, Hu H, Nobes CD, Self A, Jordens I, Goodman CS, Hall A. Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr Biol. 2001;11:339–344. [PubMed]
  • Feng L, Yunoue S, Tokuo H, Ozawa T, Zhang D, Patrakitkomjorn S, Ichimura T, Saya H, Araki N. PKA phosphorylation and 14-3-3 interaction regulate the function of neurofibromatosis type I tumor suppressor, neurofibromin. FEBS Lett. 2004;557:275–282. [PubMed]
  • Fiedler SE, Schillace RV, Daniels CJ, Andrews SF, Carr DW. Myeloid translocation gene 16b is a dual A-kinase anchoring protein that interacts selectively with plexins in a phospho-regulated manner. FEBS Lett. 2010;584:873–877. [PubMed]
  • Flavell SW, Cowan CW, Kim TK, Greer PL, Lin Y, Paradis S, Griffith EC, Hu LS, Chen C, Greenberg ME. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science. 2006;311:1008–1012. [PubMed]
  • Fukuyama T, Sueoka E, Sugio Y, Otsuka T, Niho Y, Akagi K, Kozu T. MTG8 proto-oncoprotein interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase in lymphocytes. Oncogene. 2001;20:6225–6232. [PubMed]
  • Hall A, Lalli G. Rho and Ras GTPases in axon growth, guidance, and branching. Cold Spring Harb Perspect Biol. 2010;2:a001818. [PMC free article] [PubMed]
  • Han J, Han L, Tiwari P, Wen Z, Zheng JQ. Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP. J Cell Biol. 2007;176:101–111. [PMC free article] [PubMed]
  • He H, Yang T, Terman JR, Zhang X. Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration. Proc Natl Acad Sci U S A. 2009;106:15610–15615. [PubMed]
  • Hoang B, Chiba A. Genetic analysis on the role of integrin during axon guidance in Drosophila. J Neurosci. 1998;18:7847–7855. [PubMed]
  • Hopker VH, Shewan D, Tessier-Lavigne M, Poo M, Holt C. Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1. Nature. 1999;401:69–73. [PubMed]
  • Hu H, Marton TF, Goodman CS. Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron. 2001;32:39–51. [PubMed]
  • Huang Z, Yazdani U, Thompson-Peer KL, Kolodkin AL, Terman JR. Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development. Development. 2007;134:2337–2347. [PubMed]
  • Hung RJ, Pak CW, Terman JR. Direct Redox Regulation of F-Actin Assembly and Disassembly by Mical. Science. 2011;334:1710–1713. [PMC free article] [PubMed]
  • Hung RJ, Terman JR. Extracellular inhibitors, repellents, and semaphorin/plexin/MICAL-mediated actin filament disassembly. Cytoskeleton (Hoboken) 2011;68:415–433. [PMC free article] [PubMed]
  • Hung RJ, Yazdani U, Yoon J, Wu H, Yang T, Gupta N, Huang Z, van Berkel WJ, Terman JR. Mical links semaphorins to F-actin disassembly. Nature. 2010;463:823–827. [PMC free article] [PubMed]
  • Ito Y, Oinuma I, Katoh H, Kaibuchi K, Negishi M. Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse. EMBO Rep. 2006;7:704–709. [PubMed]
  • Ivins JK, Yurchenco PD, Lander AD. Regulation of neurite outgrowth by integrin activation. J Neurosci. 2000;20:6551–6560. [PubMed]
  • Keely PJ, Rusyn EV, Cox AD, Parise LV. R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells. J Cell Biol. 1999;145:1077–1088. [PMC free article] [PubMed]
  • Keleman K, Rajagopalan S, Cleppien D, Teis D, Paiha K, Huber LA, Technau GM, Dickson BJ. Comm sorts robo to control axon guidance at the Drosophila midline. Cell. 2002;110:415–427. [PubMed]
  • Kent CB, Shimada T, Ferraro GB, Ritter B, Yam PT, McPherson PS, Charron F, Kennedy TE, Fournier AE. 14-3-3 proteins regulate protein kinase a activity to modulate growth cone turning responses. J Neurosci. 2010;30:14059–14067. [PubMed]
  • Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, Goodman CS, Tear G. Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell. 1998;92:205–215. [PubMed]
  • Kolodkin AL, Tessier-Lavigne M. Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb Perspect Biol. 2011;3 [PMC free article] [PubMed]
  • Krahn MP, Egger-Adam D, Wodarz A. PP2A antagonizes phosphorylation of Bazooka by PAR-1 to control apical-basal polarity in dividing embryonic neuroblasts. Dev Cell. 2009;16:901–908. [PubMed]
  • Mignon-Ravix C, Cacciagli P, El-Waly B, Moncla A, Milh M, Girard N, Chabrol B, Philip N, Villard L. Deletion of YWHAE in a patient with periventricular heterotopias and pronounced corpus callosum hypoplasia. J Med Genet. 2010;47:132–136. [PubMed]
  • Nagamani SC, Zhang F, Shchelochkov OA, Bi W, Ou Z, Scaglia F, Probst FJ, Shinawi M, Eng C, Hunter JV, et al. Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment. J Med Genet. 2009;46:825–833. [PubMed]
  • Nawabi H, Briancon-Marjollet A, Clark C, Sanyas I, Takamatsu H, Okuno T, Kumanogoh A, Bozon M, Takeshima K, Yoshida Y, et al. A midline switch of receptor processing regulates commissural axon guidance in vertebrates. Genes Dev. 2010;24:396–410. [PubMed]
  • Neumann S, Bradke F, Tessier-Lavigne M, Basbaum AI. Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron. 2002;34:885–893. [PubMed]
  • Nicol X, Hong KP, Spitzer NC. Spatial and temporal second messenger codes for growth cone turning. Proc Natl Acad Sci U S A. 2011;108:13776–13781. [PubMed]
  • Nozumi M, Togano T, Takahashi-Niki K, Lu J, Honda A, Taoka M, Shinkawa T, Koga H, Takeuchi K, Isobe T, et al. Identification of functional marker proteins in the mammalian growth cone. Proc Natl Acad Sci U S A. 2009;106:17211–17216. [PubMed]
  • Oinuma I, Ishikawa Y, Katoh H, Negishi M. The Semaphorin 4D receptor Plexin-B1 is a GTPase activating protein for R-Ras. Science. 2004;305:862–865. [PubMed]
  • Oinuma I, Ito Y, Katoh H, Negishi M. Semaphorin 4D/Plexin-B1 stimulates PTEN activity through R-Ras GTPase-activating protein activity, inducing growth cone collapse in hippocampal neurons. J Biol Chem. 2010;285:28200–28209. [PMC free article] [PubMed]
  • Oinuma I, Katoh H, Negishi M. Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity. J Cell Biol. 2006;173:601–613. [PMC free article] [PubMed]
  • Parra LM, Zou Y. Sonic hedgehog induces response of commissural axons to Semaphorin repulsion during midline crossing. Nat Neurosci. 2010;13:29–35. [PubMed]
  • Pearse DD, Pereira FC, Marcillo AE, Bates ML, Berrocal YA, Filbin MT, Bunge MB. cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat Med. 2004;10:610–616. [PubMed]
  • Qiu J, Cai D, Dai H, McAtee M, Hoffman PN, Bregman BS, Filbin MT. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron. 2002;34:895–903. [PubMed]
  • Rittinger K, Budman J, Xu J, Volinia S, Cantley LC, Smerdon SJ, Gamblin SJ, Yaffe MB. Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol Cell. 1999;4:153–166. [PubMed]
  • Rohm B, Rahim B, Kleiber B, Hovatta I, Puschel AW. The semaphorin 3A receptor may directly regulate the activity of small GTPases. FEBS Lett. 2000;486:68–72. [PubMed]
  • Saito Y, Oinuma I, Fujimoto S, Negishi M. Plexin-B1 is a GTPase activating protein for M-Ras, remodelling dendrite morphology. EMBO Rep. 2009;10:614–621. [PubMed]
  • Schiff M, Delahaye A, Andrieux J, Sanlaville D, Vincent-Delorme C, Aboura A, Benzacken B, Bouquillon S, Elmaleh-Berges M, Labalme A, et al. Further delineation of the 17p13.3 microdeletion involving YWHAE but distal to PAFAH1B1: four additional patients. Eur J Med Genet. 2010;53:303–308. [PubMed]
  • Schillace RV, Andrews SF, Liberty GA, Davey MP, Carr DW. Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes. J Immunol. 2002;168:1590–1599. [PubMed]
  • Self AJ, Hall A. Purification of recombinant Rho/Rac/G25K from Escherichia coli. Methods Enzymol. 1995;256:3–10. [PubMed]
  • Skoulakis EM, Davis RL. Olfactory learning deficits in mutants for leonardo, a Drosophila gene encoding a 14-3-3 protein. Neuron. 1996;17:931–944. [PubMed]
  • Skoulakis EM, Davis RL. 14-3-3 proteins in neuronal development and function. Mol Neurobiol. 1998;16:269–284. [PubMed]
  • Song H, Ming G, He Z, Lehmann M, McKerracher L, Tessier-Lavigne M, Poo M. Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science. 1998;281:1515–1518. [PubMed]
  • Stein E, Tessier-Lavigne M. Hierarchical organization of guidance receptors: silencing of netrin attraction by slit through a Robo/DCC receptor complex. Science. 2001;291:1928–1938. [PubMed]
  • Stevens A, Jacobs JR. Integrins regulate responsiveness to slit repellent signals. J Neurosci. 2002;22:4448–4455. [PubMed]
  • Su TT, Parry DH, Donahoe B, Chien CT, O’Farrell PH, Purdy A. Cell cycle roles for two 14-3-3 proteins during Drosophila development. J Cell Sci. 2001;114:3445–3454. [PMC free article] [PubMed]
  • Terman JR, Kolodkin AL. Nervy links protein kinase a to plexin-mediated semaphorin repulsion. Science. 2004;303:1204–1207. [PubMed]
  • Terman JR, Mao T, Pasterkamp RJ, Yu HH, Kolodkin AL. MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion. Cell. 2002;109:887–900. [PubMed]
  • Tong Y, Hota PK, Penachioni JY, Hamaneh MB, Kim S, Alviani RS, Shen L, He H, Tempel W, Tamagnone L, et al. Structure and function of the intracellular region of the plexin-b1 transmembrane receptor. J Biol Chem. 2009;284:35962–35972. [PMC free article] [PubMed]
  • Toyo-oka K, Shionoya A, Gambello MJ, Cardoso C, Leventer R, Ward HL, Ayala R, Tsai LH, Dobyns W, Ledbetter D, et al. 14-3-3epsilon is important for neuronal migration by binding to NUDEL: a molecular explanation for Miller-Dieker syndrome. Nat Genet. 2003;34:274–285. [PubMed]
  • Toyofuku T, Yoshida J, Sugimoto T, Zhang H, Kumanogoh A, Hori M, Kikutani H. FARP2 triggers signals for Sema3A-mediated axonal repulsion. Nat Neurosci. 2005;8:1712–1719. [PubMed]
  • Tzivion G, Shen YH, Zhu J. 14-3-3 proteins; bringing new definitions to scaffolding. Oncogene. 2001;20:6331–6338. [PubMed]
  • Uesugi K, Oinuma I, Katoh H, Negishi M. Different requirement for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1. J Biol Chem. 2009;284:6743–6751. [PMC free article] [PubMed]
  • Vikis HG, Li W, He Z, Guan KL. The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner. Proc Natl Acad Sci U S A. 2000;97:12457–12462. [PubMed]
  • Winberg ML, Mitchell KJ, Goodman CS. Genetic analysis of the mechanisms controlling target selection: complementary and combinatorial functions of netrins, semaphorins, and IgCAMs. Cell. 1998a;93:581–591. [PubMed]
  • Winberg ML, Noordermeer JN, Tamagnone L, Comoglio PM, Spriggs MK, Tessier-Lavigne M, Goodman CS. Plexin A is a neuronal semaphorin receptor that controls axon guidance. Cell. 1998b;95:903–916. [PubMed]
  • Wong W, Scott JD. AKAP signalling complexes: focal points in space and time. Nat Rev Mol Cell Biol. 2004;5:959–970. [PubMed]
  • Xu H, Leinwand SG, Dell AL, Fried-Cassorla E, Raper JA. The calmodulin-stimulated adenylate cyclase ADCY8 sets the sensitivity of zebrafish retinal axons to midline repellents and is required for normal midline crossing. J Neurosci. 2010;30:7423–7433. [PMC free article] [PubMed]
  • Yaffe MB, Elia AE. Phosphoserine/threonine-binding domains. Curr Opin Cell Biol. 2001;13:131–138. [PubMed]
  • Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, Gamblin SJ, Smerdon SJ, Cantley LC. The structural basis for 14-3-3:phosphopeptide binding specificity. Cell. 1997;91:961–971. [PubMed]
  • Yang L, Garbe DS, Bashaw GJ. A frazzled/DCC-dependent transcriptional switch regulates midline axon guidance. Science. 2009;324:944–947. [PMC free article] [PubMed]
  • Yoon BC, Zivraj KH, Strochlic L, Holt CE. 14-3-3 proteins regulate retinal axon growth by modulating ADF/cofilin activity. Dev Neurobiol 2011 [PMC free article] [PubMed]
  • Yu HH, Araj HH, Ralls SA, Kolodkin AL. The transmembrane Semaphorin Sema I is required in Drosophila for embryonic motor and CNS axon guidance. Neuron. 1998;20:207–220. [PubMed]
  • Yu L, Zhou Y, Cheng S, Rao Y. Plexin a-semaphorin-1a reverse signaling regulates photoreceptor axon guidance in Drosophila. J Neurosci. 2010;30:12151–12156. [PubMed]
  • Zhang Z, Vuori K, Wang H, Reed JC, Ruoslahti E. Integrin activation by R-ras. Cell. 1996;85:61–69. [PubMed]
  • Zhou Y, Gunput RA, Pasterkamp RJ. Semaphorin signaling: progress made and promises ahead. Trends Biochem Sci. 2008;33:161–170. [PubMed]