14-3-33 Couples Protein Kinase A
to Semaphorin Signaling and Silences
Plexin RasGAP-Mediated Axonal Repulsion
Taehong Yang1and Jonathan R. Terman1,*
1Departments of Neuroscience and Pharmacology and Neuroscience Graduate Program, The University of Texas
Southwestern Medical Center, Dallas, TX 75390, USA
The biochemical means through which multiple sig-
naling 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 silenced 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/Rap family GTPase
activating proteins (GAPs) and we find that the PlexA
GAP domain is phosphorylated by the cAMP-depen-
dent protein kinase (PKA). This PlexA phosphoryla-
tion generates a specific binding site for 14-3-33, a
phospho-binding protein that we find to be neces-
sary for axon guidance. These PKA-mediated
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
targets. Work over the past 20 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 under-
standing 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 ques-
tions 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 disrupt-
ing the adhesive interactions between an axon and its substrate
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 an actin disassembly factor, Mical (Hung et al.,
2010, 2011). 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;
Wang et al., 2012). 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 indi-
cated that Plexins exert repulsive/de-adhesive effects on
Integrin-dependent axon-substrate adhesion (Oinuma et al.,
2004, 2006; Toyofuku et al., 2005; Uesugi et al., 2009; Tong
et al., 2009; Wang et al., 2012).
Growing evidence also indicates that the repulsive effects of
axonguidance cues can besilenced and eventurned into attrac-
tion 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; Ho ¨pker et al., 1999; Dontchev and Letour-
neau, 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),
108 Neuron 74, 108–121, April 12, 2012 ª2012 Elsevier Inc.
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 repul-
sive 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.
within the Plexin GAP domain and generates a binding site for a
14-3-33. Moreover, these PKA-mediated 14-3-33-Plexin interac-
tions occlude the association between Plexin and its GAP
substrate, Ras2, concomitantly making axons less responsive
to Sema-mediated repulsion and more responsive to Integrin-
mediated adhesion. Our findings, therefore, uncover both a
molecular integration point between important axon guidance
signaling pathways and a biochemical logic by which this guid-
ance information is coalesced to steer the growing axon.
14-3-33 Is Neuronal PlexA-Interacting Protein
The C-terminal region of the 14-3-33 protein was identified as
a strong Drosophila Plexin A (PlexA) interactor in a yeast two-
hybrid interaction screen (Figures 1Aa–1Ac). 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), 14-3-33,
and 14-3-3z/leonardo (Figure S1A available online), but PlexA
selectively interacted with only 14-3-33 in our yeast interaction
assay (Figure S1B). Likewise, we saw selective interactions
between neuronally expressedHAPlexA and purified recombi-
nant GST-14-3-33 protein (Figure 1Ad). The other Drosophila
Plexin, PlexB, did not interact with 14-3-33 in our yeast interac-
tion assay (Figure 1Ac), also suggesting a specificity among
Figure 1. 14-3-33 Is a Neuronal PlexA Interacting Protein
(A) 14-3-33 is a PlexA-specific interacting protein. The highly conserved Cyto2 region (amino acid [aa] residues 1702-1945) of PlexA (a) was the bait in a yeast
two-hybrid screen and interacts with Clone 135, which encodes aa98-261 of 14-3-33 (b), as measured by Beta-galactosidase (Beta Gal) activity (c). PlexACyto2
also interacts with the related clone 135* (aa97-261). No interactions are observed between PlexBCyto2and Clone 135 but Clone 135 also strongly interacts with
another form of PlexA (PlexACyto2 Trunc). Likewise, purified GST-14-3-33 protein, but not beads or GST protein only, added to Drosophila embryonic lysates
robustly associates and pulls downneuronally expressedHAPlexA(d).Equal amountsofpurifiedGST-tagged proteins (Pull-down,Coomassie)and lysates(Input)
were added. Molecular weight (Mw) in kDa. The interaction compared to that of beads only was quantified. n = 4; error bar: SEM; *p < 0.05 by paired t test.
(B) 14-3-33 associates with PlexA in neurons. 14-3-33 (a) and PlexA (b) transcripts are both highly enriched in the Drosophila embryonic CNS (brain and cord).
Likewise, 14-3-33 protein (c, c0, c00) is highly expressed in the developing nervous system and localizes to CNS (longitudinal [L], commissural [arrowhead]) and
motor (arrows) axons. The boxed region in (c) is seen at higher power in c0and likewise for c0in c00. PlexA and 14-3-33 also associate in vivo in neurons (d).
Embryonic lysates from Drosophila embryos expressingHAPlexA andFLAG14-3-33 in neurons were subjected to immunoprecipitation with antibodies against HA.
Antibodies againstHAPlexA (top) immunoprecipitateFLAG14-3-33 (bottom) while controls (beads only or IgG antibody) do not.
See also Figure S1.
PKA, 14-3-33, and Semaphorin-Plexin Axon Repulsion
Neuron 74, 108–121, April 12, 2012 ª2012 Elsevier Inc. 109
Hu, H., Marton, T.F., and Goodman, C.S. (2001). Plexin B mediates axon guid-
ance in Drosophila by simultaneously inhibiting active Rac and enhancing
RhoA signaling. Neuron 32, 39–51.
Huang, Z., Yazdani, U., Thompson-Peer, K.L., Kolodkin, A.L., and Terman,
J.R. (2007). Crk-associated substrate (Cas) signaling protein functions with in-
tegrins to specify axon guidance during development. Development 134,
Hung, R.J., and Terman, J.R. (2011). Extracellular inhibitors, repellents, and
semaphorin/plexin/MICAL-mediated actin filament disassembly. Cytoskeleton
Hung, R.J., Yazdani, U., Yoon, J., Wu, H., Yang, T., Gupta, N., Huang, Z., van
Berkel, W.J., and Terman, J.R. (2010). Mical links semaphorins to F-actin
disassembly. Nature 463, 823–827.
Hung, R.J., Pak, C.W., and Terman, J.R. (2011). Direct redox regulation of
F-actin assembly and disassembly by Mical. Science 334, 1710–1713.
Ito, Y., Oinuma, I., Katoh, H., Kaibuchi, K., and Negishi, M. (2006). Sema4D/
plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth
cone collapse. EMBO Rep. 7, 704–709.
Ivins, J.K., Yurchenco, P.D., and Lander, A.D. (2000). Regulation of neurite
outgrowth by integrin activation. J. Neurosci. 20, 6551–6560.
Keely, P.J., Rusyn, E.V., Cox, A.D., and Parise, L.V. (1999). R-Ras signals
through specific integrin alpha cytoplasmic domains to promote migration
and invasion of breast epithelial cells. J. Cell Biol. 145, 1077–1088.
Keleman, K., Rajagopalan, S., Cleppien, D., Teis, D., Paiha, K., Huber, L.A.,
Technau, G.M., and Dickson, B.J. (2002). Comm sorts robo to control axon
guidance at the Drosophila midline. Cell 110, 415–427.
Kent, C.B., Shimada, T., Ferraro, G.B., Ritter, B., Yam, P.T., McPherson, P.S.,
Charron, F., Kennedy, T.E., and Fournier, A.E. (2010). 14-3-3 proteins regulate
protein kinase a activity to modulate growth cone turning responses.
J. Neurosci. 30, 14059–14067.
Kidd, T., Brose, K., Mitchell, K.J., Fetter, R.D., Tessier-Lavigne, M., Goodman,
C.S., and Tear, G. (1998). Roundabout controls axon crossing of the CNS
midline and defines a novel subfamily of evolutionarily conserved guidance
receptors. Cell 92, 205–215.
Kolodkin, A.L., and Tessier-Lavigne, M. (2011). Mechanisms and molecules of
neuronal wiring: a primer. Cold Spring Harb. Perspect. Biol. 3, a001727.
phorylation of Bazooka by PAR-1 to control apical-basal polarity in dividing
embryonic neuroblasts. Dev. Cell 16, 901–908.
Mignon-Ravix, C., Cacciagli, P., El-Waly, B., Moncla, A., Milh, M., Girard, N.,
Chabrol, B., Philip, N., and Villard, L. (2010). Deletion of YWHAE in a patient
with periventricular heterotopias and pronounced corpus callosum hypo-
plasia. J. Med. Genet. 47, 132–136.
Nagamani, S.C., Zhang, F., Shchelochkov, O.A., Bi, W., Ou, Z., Scaglia, F.,
Probst, F.J., Shinawi, M., Eng, C., Hunter, J.V., et al. (2009). 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. 46, 825–833.
Nawabi, H., Brianc ¸on-Marjollet, A., Clark, C., Sanyas, I., Takamatsu, H.,
Okuno, T., Kumanogoh, A., Bozon, M., Takeshima, K., Yoshida, Y., et al.
(2010). A midline switch of receptor processing regulates commissural axon
guidance in vertebrates. Genes Dev. 24, 396–410.
Neumann, S., Bradke, F., Tessier-Lavigne, M., and Basbaum, A.I. (2002).
Regeneration of sensory axons within the injured spinal cord induced by intra-
ganglionic cAMP elevation. Neuron 34, 885–893.
Nicol, X., Hong, K.P., and Spitzer, N.C. (2011). Spatial and temporal second
messenger codes for growth cone turning. Proc. Natl. Acad. Sci. USA 108,
Nozumi, M., Togano, T., Takahashi-Niki, K., Lu, J., Honda, A., Taoka, M.,
Shinkawa, T., Koga, H., Takeuchi, K., Isobe, T., and Igarashi, M. (2009).
Identification of functional marker proteins in the mammalian growth cone.
Proc. Natl. Acad. Sci. USA 106, 17211–17216.
Oinuma,I.,Ishikawa, Y.,Katoh,H.,andNegishi,M.(2004).TheSemaphorin 4D
receptor Plexin-B1 is a GTPase activating protein for R-Ras. Science 305,
ated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin
activity. J. Cell Biol. 173, 601–613.
stimulates PTEN activity through R-Ras GTPase-activating protein activity,
inducing growth cone collapse in hippocampal neurons. J. Biol. Chem. 285,
Parra, L.M., and Zou, Y. (2010). Sonic hedgehog induces response of commis-
sural axons to Semaphorin repulsion during midline crossing. Nat. Neurosci.
Pearse, D.D., Pereira, F.C., Marcillo, A.E., Bates, M.L., Berrocal, Y.A., Filbin,
M.T., and Bunge, M.B. (2004). cAMP and Schwann cells promote axonal
growth and functional recovery after spinal cord injury. Nat. Med. 10, 610–616.
Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P.N., Bregman, B.S., and Filbin,
M.T. (2002). Spinal axon regeneration induced by elevation of cyclic AMP.
Neuron 34, 895–903.
Rittinger, K., Budman, J., Xu, J., Volinia, S., Cantley, L.C., Smerdon, S.J.,
Gamblin, S.J., and Yaffe, M.B. (1999). Structural analysis of 14-3-3 phospho-
peptide complexes identifies a dual role for the nuclear export signal of 14-3-3
in ligand binding. Mol. Cell 4, 153–166.
Rohm, B., Rahim, B., Kleiber, B., Hovatta, I., and Pu ¨schel, A.W. (2000). The
semaphorin 3A receptor may directly regulate the activity of small GTPases.
FEBS Lett. 486, 68–72.
Saito, Y., Oinuma, I., Fujimoto, S., and Negishi, M. (2009). Plexin-B1 is
a GTPase activating protein for M-Ras, remodelling dendrite morphology.
EMBO Rep. 10, 614–621.
Schiff, M., Delahaye, A., Andrieux, J., Sanlaville, D., Vincent-Delorme, C.,
Aboura, A., Benzacken, B., Bouquillon, S., Elmaleh-Berges, M., Labalme, A.,
et al. (2010). Further delineation of the 17p13.3 microdeletion involving
YWHAE but distal to PAFAH1B1: four additional patients. Eur. J. Med.
Genet. 53, 303–308.
Schillace, R.V., Andrews, S.F., Liberty, G.A., Davey, M.P., and Carr, D.W.
(2002). Identification and characterization of myeloid translocation gene 16b
as a novel a kinase anchoring protein in T lymphocytes. J. Immunol. 168,
Self, A.J., and Hall, A. (1995). Purification of recombinant Rho/Rac/G25K from
Escherichia coli. Methods Enzymol. 256, 3–10.
Skoulakis, E.M., and Davis, R.L. (1996). Olfactory learning deficits in mutants
for leonardo, a Drosophila gene encoding a 14-3-3 protein. Neuron 17,
Skoulakis, E.M., and Davis, R.L. (1998). 14-3-3 proteins in neuronal develop-
ment and function. Mol. Neurobiol. 16, 269–284.
Song, H., Ming, G., He, Z., Lehmann, M., McKerracher, L., Tessier-Lavigne,
M., and Poo, M. (1998). Conversion of neuronal growth cone responses from
repulsion to attraction by cyclic nucleotides. Science 281, 1515–1518.
Stein, E., and Tessier-Lavigne, M. (2001). Hierarchical organization of guid-
ance receptors: silencing of netrin attraction by slit through a Robo/DCC
receptor complex. Science 291, 1928–1938.
Stevens, A., and Jacobs, J.R. (2002). Integrins regulate responsiveness to slit
repellent signals. J. Neurosci. 22, 4448–4455.
Su, T.T., Parry, D.H., Donahoe, B., Chien, C.T., O’Farrell, P.H., and Purdy, A.
J. Cell Sci. 114, 3445–3454.
Terman, J.R., and Kolodkin, A.L. (2004). Nervy links protein kinase a to plexin-
mediated semaphorin repulsion. Science 303, 1204–1207.
Terman, J.R., Mao, T., Pasterkamp, R.J., Yu, H.H., and Kolodkin, A.L. (2002).
MICALs, a family of conserved flavoprotein oxidoreductases, function in
plexin-mediated axonal repulsion. Cell 109, 887–900.
PKA, 14-3-33, and Semaphorin-Plexin Axon Repulsion
120 Neuron 74, 108–121, April 12, 2012 ª2012 Elsevier Inc.
Tong, Y., Hota, P.K., Penachioni, J.Y., Hamaneh, M.B., Kim, S., Alviani, R.S.,
Shen, L., He, H., Tempel, W., Tamagnone, L., et al. (2009). Structure and
function of the intracellular region of the plexin-b1 transmembrane receptor.
J. Biol. Chem. 284, 35962–35972.
Toyo-oka, K., Shionoya, A., Gambello, M.J., Cardoso, C., Leventer, R.,
Ward, H.L., Ayala, R., Tsai, L.H., Dobyns, W., Ledbetter, D., et al. (2003).
14-3-3epsilon is important for neuronal migration by binding to NUDEL:
a molecular explanation for Miller-Dieker syndrome. Nat. Genet. 34, 274–285.
Toyofuku, T., Yoshida, J., Sugimoto, T., Zhang, H., Kumanogoh, A., Hori, M.,
and Kikutani, H. (2005). FARP2 triggers signals for Sema3A-mediated axonal
repulsion. Nat. Neurosci. 8, 1712–1719.
Tzivion, G., Shen, Y.H., and Zhu, J. (2001). 14-3-3 proteins; bringing new
definitions to scaffolding. Oncogene 20, 6331–6338.
Uesugi, K., Oinuma,I., Katoh, H., and Negishi, M. (2009). Different requirement
for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1. J. Biol.
Chem. 284, 6743–6751.
Vikis, H.G., Li, W., He, Z., and Guan, K.L. (2000). The semaphorin receptor
plexin-B1 specifically interacts with active Rac in a ligand-dependent manner.
Proc. Natl. Acad. Sci. USA 97, 12457–12462.
Wang, Y., He, H., Srivastava, N., Vikarunnessa, S., Chen, Y.B., Jiang, J.,
Cowan, C.W., and Zhang, X. (2012). Plexins are GTPase-activating proteins
for Rap and are activated by induced dimerization. Sci. Signal. 5, ra6.
Winberg, M.L., Mitchell, K.J., and Goodman, C.S. (1998a). Genetic analysis of
the mechanisms controlling target selection: complementary and combinato-
rial functions of netrins, semaphorins, and IgCAMs. Cell 93, 581–591.
Winberg, M.L., Noordermeer, J.N., Tamagnone, L., Comoglio, P.M., Spriggs,
M.K., Tessier-Lavigne, M., and Goodman, C.S. (1998b). Plexin A is a neuronal
semaphorin receptor that controls axon guidance. Cell 95, 903–916.
Wong, W., and Scott, J.D. (2004). AKAP signalling complexes: focal points in
space and time. Nat. Rev. Mol. Cell Biol. 5, 959–970.
Xu, H., Leinwand, S.G., Dell, A.L., Fried-Cassorla, E., and Raper, J.A. (2010).
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. 30, 7423–7433.
Yaffe, M.B., and Elia, A.E. (2001). Phosphoserine/threonine-binding domains.
Curr. Opin. Cell Biol. 13, 131–138.
Yaffe, M.B., Rittinger, K., Volinia, S., Caron, P.R., Aitken, A., Leffers, H.,
Gamblin, S.J., Smerdon, S.J., and Cantley, L.C. (1997). The structural basis
for 14-3-3:phosphopeptide binding specificity. Cell 91, 961–971.
Yang, L., Garbe, D.S., and Bashaw, G.J. (2009). A frazzled/DCC-dependent
Yoon, B.C., Zivraj, K.H., Strochlic, L., and Holt, C.E. (2011). 14-3-3 proteins
regulate retinal axon growth by modulating ADF/cofilin activity. Dev.
Yu, H.H., Araj, H.H., Ralls, S.A., and Kolodkin, A.L.(1998). The transmembrane
Semaphorin Sema I is required in Drosophila for embryonic motor and CNS
axon guidance. Neuron 20, 207–220.
Yu, L., Zhou, Y., Cheng, S., and Rao, Y. (2010). Plexin a-semaphorin-1a
reverse signaling regulates photoreceptor axon guidance in Drosophila.
J. Neurosci. 30, 12151–12156.
Zhang, Z., Vuori, K., Wang, H., Reed, J.C., and Ruoslahti, E. (1996). Integrin
activation by R-ras. Cell 85, 61–69.
Zhou, Y., Gunput, R.A., and Pasterkamp, R.J. (2008). Semaphorin signaling:
progress made and promises ahead. Trends Biochem. Sci. 33, 161–170.
PKA, 14-3-33, and Semaphorin-Plexin Axon Repulsion
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