Plexin-B1/RhoGEF-mediated RhoA activation involves the receptor tyrosine kinase ErbB-2

Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2-mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse.     

Interaction of plexin-B1 with PDZ domain-containing Rho guanine nucleotide exchange factors

The Rho family GTPase has been implicated in plexin-B1, a receptor for Semaphorin 4D (Sema4D), mediating signal transduction. Rho may also play a function in this signaling pathway as well as Rac, but the mechanisms for Rho regulation are poorly understood. In this study, we have identified two kinds of PDZ domain-containing Rho-specific guanine nucleotide exchange factors (RhoGEFs) as proteins interacting with plexin-B1 cytoplasmic domain. These PDZ domain-containing RhoGEFs showed significant homology to human KIAA0380 (PDZ-RhoGEF) and LARG (KIAA0382), respectively. Both KIAA0380 and LARG could bind plexin-B1 and a deletion mutant analysis of plexin-B1, KIAA0380 and LARG revealed that KIAA0380 and LARG bound plexin-B1 cytoplasmic tail through their PDZ domains. The tissue distribution analysis indicated that plexin-B1 was co-localized with KIAA0380 and LARG in various tissues. Immunocytochemical analysis showed that LARG was recruited to plasma membrane by plexin-B1. These results suggest that PDZ domain-containing RhoGEFs play a role in Sema4D-plexin-B1 mediating signal transduction.     

Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF

Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. Here we show that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, we show that ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration.     

Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). Here we characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppressed R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressed the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocked the growth cone collapse. The R-Ras GAP activity was necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and was also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induced dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. We conclude that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.     

B plexins activate Rho through PDZ-RhoGEF

Plexins are receptors for the repulsive axon guidance molecules semaphorins. Previously, we have shown that plexin-B1 binds activated Rac, but that clustering of plexin-B1 causes Rho activation, resulting in stress fiber formation. Using the yeast two-hybrid system, we found that the C-terminus of B plexins interacted directly with Rho-specific exchange factors, via their PDZ domain. Mutation of the carboxy-terminal amino acids of plexin-B1 or coexpression of a dominant negative PDZ-RhoGEF abrogated the ability of plexin-B1 to cause stress fiber formation. Our results demonstrate a role for PDZ-RhoGEF in B plexin-mediated activation of Rho/Rho kinase signaling, implicated in the regulation of axon guidance and cell migration.     

Semaphorin 4D Signaling Requires the Recruitment of Phospholipase Cγ into the Plexin-B1 Receptor Complex

The semaphorin 4D (Sema4D) receptor plexin-B1 constitutively interacts with particular Rho guanine nucleotide exchange factors (RhoGEFs) and thereby mediates Sema4D-induced RhoA activation, a process which involves the tyrosine phosphorylation of plexin-B1 by ErbB-2. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGEF activity. We show here that activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation creates docking sites for the SH2 domains of phospholipase Cγ (PLCγ). PLCγ is thereby recruited into the plexin-B1 receptor complex and via its SH3 domain activates the Rho guanine nucleotide exchange factor PDZ-RhoGEF. PLCγ-dependent RhoGEF activation is independent of its lipase activity. The recruitment of PLCγ has no effect on the R-Ras GTPase-activating protein activity of plexin-B1 but is required for Sema4D-induced axonal growth cone collapse as well as for the promigratory effects of Sema4D on cancer cells. These data demonstrate a novel nonenzymatic function of PLCγ as an important mechanism of plexin-mediated signaling which links tyrosine phosphorylation of plexin-B1 to the regulation of a RhoGEF protein and downstream cellular processes.Mammalian semaphorins were originally identified as axon guidance factors but are now recognized also as important regulators of morphogenesis and homeostasis in various organ systems, including the immune, cardiovascular, and renal systems (3-5, 7, 19, 23, 30, 35, 40, 56, 64, 76). Most effects of semaphorins are mediated by a group of large transmembrane proteins called plexins, of which four families exist in the mammalian system: plexin-A1 to -4, plexin-B1 to -3, plexin-C1, and plexin-D1 (60, 61). The four members of the plexin-A family in most cases require neuropilins as ligand binding partners to respond to semaphorins, whereas the three members of the plexin-B family are directly activated by semaphorins. While plexin-B1 binds Sema4D, plexin-B2 can be activated by Sema4C and Sema4D, and plexin-B3 has been shown to respond to Sema5A (31, 35).The activation of plexins by semaphorins initiates a variety of signaling processes, which involve several small GTPases of the Ras and Rho families (31, 34, 43). All plexin family members possess an R-Ras GTPase-activating protein (GAP) domain (36). Activated plexin-B1 and -A1 have been shown to also interact with other small GTPases, including GTP-bound Rac1 and RhoD as well as Rnd1, Rnd2, and Rnd3 (14, 37, 48, 63, 67, 68, 74). Different from other plexin families, the C terminus of B-family plexins contains a PDZ domain-binding motif which mediates a stable interaction with the guanine nucleotide exchange factors PDZ-RhoGEF and LARG (1, 15, 26, 39, 57). Activation of the plexin-B1/PDZ-RhoGEF complex by semaphorin 4D (Sema4D) results in RhoA activation downstream of plexin-B1 (15, 39, 57). Members of the plexin-B family also interact with and are phosphorylated by the receptor tyrosine kinases ErbB-2 and c-Met (12, 22, 58). ErbB-2-mediated phosphorylation of plexin-B1 is required for plexin-mediated RhoA activation and downstream cellular effects, including the promigratory effects of Sema4D on cancer cells and the induction of axonal growth cone collapse by Sema4D (58, 59). However, the molecular mechanisms linking ErbB-2-mediated phosphorylation of plexin-B1 to the regulation of RhoA activity and subsequent cellular effects are unknown.Here we report that upon activation by Sema4D, plexin-B1 becomes phosphorylated by ErbB-2 at particular tyrosine residues on its intracellular portion. These phosphorylated tyrosine residues serve as docking sites for the SH2 domains of PLCγ. PLCγ is thereby recruited into the plexin-B1 receptor complex and through its SH3 domain mediates RhoA activation and downstream cellular effects.     

Control of semaphorin signaling

Receptor complexes for the chemorepellent factors of the semaphorin family activate intracellular pathways that trigger actin rearrangements underlying growth cone collapse and repellent behavior. Some evidence has been provided for a complex and dynamic pattern of interaction between members of the small Rho guanosine triphosphatases and plexin proteins that are the receptor subunits responsible for initiating semaphorin signaling. The characterization of new components of semaphorin receptor complexes, the implication of several distinct classes of cytoplasmic effectors, together with the observation of a variety of processes modulating the semaphorin signal have provided a basis for a much improved, but still intricate view of the semaphorin transduction pathways in neurons.     

Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells

Plexins are receptors for the axon guidance molecule semaphorins, and several lines of evidence suggest that Rho family small GTPases are implicated in the downstream signaling of Plexins. Recent studies have demonstrated that Plexin-B1 activates RhoA and induces growth cone collapse through Rho-specific guanine nucleotide exchange factor PDZ-RhoGEF. Here we show that Rnd1, a member of Rho family GTPases, directly interacted with the cytoplasmic domain of Plexin-B1. In COS-7 cells, coexpression of Rnd1 and Plexin-B1 induced cell contraction in response to semaphorin 4D (Sema4D), a ligand for Plexin-B1, whereas expression of Plexin-B1 alone or coexpression of Rnd1 and a Rnd1 interaction-defective mutant of Plexin-B1 did not. The Sema4D-induced contraction in Plexin-B1/Rnd1-expressing COS-7 cells was suppressed by dominant negative RhoA, a Rho-associated kinase inhibitor, a dominant negative form of PDZ-RhoGEF, or deletion of the carboxyl-terminal PDZ-RhoGEF-binding region of Plexin-B1, indicating that the PDZ-RhoGEF/RhoA/Rho-associated kinase pathway is involved in this morphological effect. We also found that Rnd1 promoted the interaction between Plexin-B1 and PDZ-RhoGEF and thereby dramatically potentiated the Plexin-B1-mediated RhoA activation. We propose that Rnd1 plays an important role in the regulation of Plexin-B1 signaling, leading to Rho activation during axon guidance and cell migration.     

Molecular basis of semaphorin-mediated axon guidance

The semaphorin family of proteins constitute one of the major cues for axonal guidance. The prototypic member of this family is Sema3A, previously designated semD/III or collapsin-1. Sema3A acts as a diffusible, repulsive guidance cue in vivo for the peripheral projections of embryonic dorsal root ganglion neurons. Sema3A binds with high affinity to neuropilin-1 on growth cone filopodial tips. Although neuropilin-1 is required for Sema3A action, it is incapable of transmitting a Sema3A signal to the growth cone interior. Instead, the Sema3A/neuropilin-1 complex interacts with another transmembrane protein, plexin, on the surface of growth cones. Certain semaphorins, other than Sema3A, can bind directly to plexins. The intracellular domain of plexin is responsible for initiating the signal transduction cascade leading to growth cone collapse, axon repulsion, or growth cone turning. This intracellular cascade involves the monomeric G-protein, Rac1, and a family of neuronal proteins, the CRMPs. Rac1 is likely to be involved in semaphorin-induced rearrangements of the actin cytoskeleton, but how plexin controls Rac1 activity is not known. Vertebrate CRMPs are homologous to the Caenorhabditis elegans unc-33 protein, which is required for proper axon morphology in worms. CRMPs are essential for Sema3A-induced, neuropilin-plexin-mediated growth cone collapse, but the molecular interactions of growth cone CRMPs are not well defined. Mechanistic aspects of plexin-based signaling for semaphorin guidance cues may have implications for other axon guidance events and for the basis of growth cone motility.     

Plexin-B3 is a functional receptor for semaphorin 5A

Semaphorins are a large family of molecular cues implicated in neural development and in a variety of functions outside the nervous system. Semaphorin 5A (Sema5A) is a transmembrane semaphorin, containing seven thrombospondin type-1 repeats, which was recently found to control axon guidance. Here we show that plexin-B3 is a high-affinity receptor specific for Sema5A. We further demonstrate that plexin-B3 activation by Sema5A mediates functional responses in plexin-B3-expressing cells (either fibroblasts, epithelial and primary endothelial cells). In addition, Sema5A can trigger the intracellular signalling of the hepatocyte growth factor/scatter factor receptor, Met, associated in a complex with plexin-B3. We thus conclude that Sema5A is able to elicit multiple functional responses through its receptor plexin-B3.     

The activity of the plexin-A1 receptor is regulated by Rac

Plexins constitute a large family of transmembrane proteins that act as receptors for the semaphorin family of ligands. They are best known for their role in growth cone guidance, although they also are widely expressed outside the nervous system. Plexins are thought to control axon guidance by modifying the growth cone cytoskeleton, and Rho GTPases have been strongly implicated in this response. However, the exact contribution of Rho proteins is unclear. Sema3A/Plexin-A1-induced growth cone collapse, for example, requires Rac activity, which is a surprising result given that this GTPase is usually associated with membrane protrusions. We show here that Sema3A-induced collapse of COS-7 cells expressing Plexin-A1 also requires Rac but not Rho activity and that the cytoplasmic tail of Plexin-A1 interacts directly with activated Rac. However, collapse induced by a constitutively activated version of Plexin-A1 does not require Rac. We propose a novel function for Rac, namely that it acts upstream of Plexin-A1 during semaphoring-induced collapse, to regulate the activity of the receptor.     

Growth cone neuropilin-1 mediates collapsin-1/Sema III facilitation of antero- and retrograde axoplasmic transport.

Collapsin-1/Sema III, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin-1 exerts its action are not fully understood. Collapsin-1 induces growth cone collapse via a pathway which may include neuropilin-1, a cellsurface collapsin-1 binding protein, as well as intracellular CRMP-62 and heterotrimeric G proteins. We previously identified a second action of collapsin-1, the facilitation of antero- and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin-1 in the action of collapsin-1 on axoplasmic transport, we produced a soluble neuropilin-1 (sNP-1) lacking the transmembrane and intracellular region. sNP-1 progressively displaced the dose-response curve for collapsin-1 to induce growth cone collapse to higher concentrations. sNP-1 also inhibited collapsin-1-induced augmentation of both antero- and retrograde axoplasmic transport. Furthermore, an anti-neuropilin-1 antibody blocked the collapsin-induced axoplasmic transport. These results together indicate that neuropilin-1 mediates collapsin-1 action on axoplasmic transport. To visualize collapsin-1 binding to endogenous neuropilin-1, we used a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4). CAP-4 stains the growth cone, neurite, and cell body. However, local application of collapsin-1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP-1 mediates the facilitatory action of collapsin-1 on antero- and retrograde axoplasmic transport.     

CHL1 promotes Sema3A-induced growth cone collapse and neurite elaboration through a motif required for recruitment of ERM proteins to the plasma membrane

Close homolog of L1 (CHL1) is a transmembrane cell adhesion molecule with unique developmental functions in cortical neuronal positioning and dendritic projection within the L1 family, as well as shared functions in promotion of integrin-dependent neurite outgrowth and semaphorin3A (Sema3A)-mediated axon repulsion. The molecular mechanisms by which CHL1 mediates these diverse functions are obscure. Here it is demonstrated using a cytofluorescence assay that CHL1 is able to recruit ezrin, a member of the ezrin-radixin-moesin (ERM) family of filamentous actin binding proteins to the plasma membrane, and that this requires a membrane-proximal motif (RGGKYSV) in the CHL1 cytoplasmic domain. This sequence in CHL1 is shown to have novel functions necessary for Sema3A-induced growth cone collapse and CHL1-dependent neurite outgrowth and branching in cortical embryonic neurons. In addition, stimulation of haptotactic cell migration and cellular adhesion to fibronectin by CHL1 depends on the CHL1/ERM recruitment motif. These findings suggest that a direct or indirect interaction between CHL1 and ERM proteins mediates Sema3A-induced growth cone collapse as well as neurite outgrowth and branching, which are essential determinants of axon guidance and connectivity in cortical development.     

Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G alpha 12 and G alpha 13 but also with G alpha q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G alpha q, G alpha 12, and G alpha 13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G alpha q and G alpha 13. Activated G alpha q, as well as G alpha 12 and G alpha 13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G alpha subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.     

Unc5B associates with LARG to mediate the action of repulsive guidance molecule

Neuronal axons are guided by attractive and repulsive cues in their local environment. Because the repulsive guidance molecule A (RGMa) was originally identified as an axon repellent in the visual system, diverse functions in the developing and adult central nervous system have been ascribed to it. RGMa binding to its receptor neogenin induces RhoA activation, leading to inhibitory/repulsive behavior and collapse of the neuronal growth cone. However, the precise mechanisms that regulate RhoA activation are poorly understood. In this study, we show that Unc5B, a member of the netrin receptor family, interacts with neogenin as a coreceptor for RGMa. Moreover, leukemia-associated guanine nucleotide exchange factor (LARG) associates with Unc5B to transduce the RhoA signal. Focal adhesion kinase (FAK) is involved in RGMa-induced tyrosine phosphorylation of LARG as well as RhoA activation. These findings uncover the molecular basis for diverse functions mediated by RGMa.     

Growth cone neuropilin‐1 mediates collapsin‐1/sema III facilitation of antero‐ and retrograde axoplasmic transport

Collapsin‐1/SemaIII, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin‐1 exerts its action are not fully understood. Collapsin‐1 induces growth cone collapse via a pathway which may include neuropilin‐1, a cell‐surface collapsin‐1 binding protein, as well as intracellular CRMP‐62 and heterotrimeric G proteins. We previously identified a second action of collapsin‐1, the facilitation of antero‐ and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin‐1 in the action of collapsin‐1 on axoplasmic transport, we produced a soluble neuropilin‐1 (sNP‐1) lacking the transmembrane and intracellular region. sNP‐1 progressively displaced the dose–response curve for collapsin‐1 to induce growth cone collapse to higher concentrations. sNP‐1 also inhibited collapsin‐1‐induced augmentation of both antero‐ and retrograde axoplasmic transport. Furthermore, an anti‐neuropilin‐1 antibody blocked the collapsin‐induced axoplasmic transport. These results together indicate that neuropilin‐1 mediates collapsin‐1 action on axoplasmic transport. To visualize collapsin‐1 binding to endogenous neuropilin‐1, we used a truncated collapsin‐1–alkaline phosphatase fusion protein (CAP‐4). CAP‐4 stains the growth cone, neurite, and cell body. However, local application of collapsin‐1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP‐1 mediates the facilitatory action of collapsin‐1 on antero‐ and retrograde axoplasmic transport. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 579–589, 1999     

R-Ras fills another GAP in semaphorin signalling

Plexins are cell-surface receptors for the semaphorin family of neuronal guidance cues. Following semaphorin binding, the plexin cytoplasmic region initiates poorly understood signal-transduction events that lead to modifications of the cytoskeleton. Recent findings shed new light on the signalling network downstream of semaphorins and plexins by demonstrating that one of the plexins, plexin-B1, possesses an intrinsic GTPase-activating protein (GAP) activity towards R-Ras. Inactivation of R-Ras by the plexin-B1 GAP domains is required for plexin-B1-mediated effects on the cytoskeleton. These results indicate that plexins not only bind to but also regulate directly the activity of some of their downstream effectors.     

Collapsin response mediator protein-4 regulates F-actin bundling

Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.     

Semaphorin 3A inhibits ERM protein phosphorylation in growth cone filopodia through inactivation of PI3K

Ezrin-radixin-moesin (ERM) proteins are involved in the linkage of membranes to theactin filament (F-actin) cytoskeleton. Phosphorylation of the C-terminus activates the F-actin binding domain of ERM proteins by preventing the action of an autoinhibitory domain. In this study, we investigated whether a growth cone collapsing signal, semaphorin 3A (Sema3A), alters the state of ERM C-terminus phosphorylation. In the growth cones of dorsal root ganglion axons, phosphorylated ERM proteins localize to filopodia. We report that Sema3A inhibits ERM protein phosphorylation in growth cone filopodia. Significantly, Sema3A decreased ERM phosphorylation prior to the onset of growth cone collapse. Over-expression of the F-actin binding fragment of ERM proteins, which competes with endogenous ERM proteins for binding to F-actin, inhibited filopodial initiation and dynamics. Sema3A has been previously shown to inhibit phosphoinositide 3-kinase (PI3K) activity. Inhibition of PI3K resulted in the loss of phosphorylated ERM proteins from growth cone filopodia, and treatment with a PI3K activating peptide blocked the effects of Sema3A on ERM phosphorylation. Collectively, these observations demonstrate that inactivation of PI3K in response to Sema3A results in decreased phosphorylation of ERM proteins in filopodia thereby contributing to growth cone collapse.     

Semaphorin 4D/Plexin-B1 Stimulates PTEN Activity through R-Ras GTPase-activating Protein Activity,Inducing Growth Cone Collapse in Hippocampal Neurons

Plexins are receptors for axonal guidance molecules semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). Here we examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. We also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 α activity. Thus, we propose that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse.