Izumi Oinuma

Molecular Dissection of the Semaphorin 4D Receptor Plexin-B1-Stimulated R-Ras GTPase-Activating Protein Activity and Neurite Remodeling in Hippocampal Neurons

Plexins serve as receptors for repulsive axonal guidance molecules semaphorins. The cytoplasmic domain of the semaphorin 4D (Sema4D) receptor, Plexin-B1 has two separated Ras GTPase-activating protein (GAP)-homologous domains, C1 and C2. Recently, we reported that the Rho family small GTPase Rnd1 associates with Plexin-B1, and the Plexin-B1-Rnd1 complex stimulates GTPase activity of R-Ras, inducing growth cone collapse in hippocampal neurons in response to Sema4D. However, the molecular mechanisms by which Plexin-B1 exhibits the GAP activity remain unclear. In this report, critical roles of Rnd1 and Sema4D in Plexin-B1-stimulated R-Ras GAP activity and neurite remodeling were examined. The N-terminal region of the cytoplasmic domain of Plexin-B1 containing the C1 domain interacts with the C-terminal region containing the C2 domain, and Rnd1 disrupts this interaction. On the other hand, Sema4D induces clustering of Rnd1-bound Plexin-B1, in parallel with inactivation of R-Ras in cells. Antibody clustering of the recombinant cytoplasmic domain of Plexin-B1 in the presence of Rnd1 triggers the R-Ras GAP activity. Deletion of the extracellular domain of Plexin-B1 causes ligand-independent clustering of the receptor, rendering the receptor constitutively active in the presence of Rnd1, and induces contraction of COS-7 cells and inhibition of neurite outgrowth in hippocampal neurons. These results indicate that Rnd1 opens the two R-Ras GAP domains of Plexin-B1, and Sema4D-induced receptor clustering stimulates R-Ras GAP activity and neurite remodeling in hippocampal neurons.

Sema4D/plexin‐B1 activates GSK‐3β 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)‐3β. 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‐3β, also blocked the growth cone collapse. The R‐Ras GAP activity was necessary for plexin‐B1‐induced dephosphorylation of Akt and activation of GSK‐3β and was also required for phosphorylation of a downstream kinase of GSK‐3β, collapsin response mediator protein‐2. Plexin‐A1 also induced dephosphorylation of Akt and GSK‐3β through its R‐Ras GAP activity. We conclude that plexin‐B1 inactivates PI(3)K and dephosphorylates Akt and GSK‐3β through R‐Ras GAP activity, inducing growth cone collapse.

Plexins: axon guidance and signal transduction

Abstract.Axon guidance represents a key stage in the formation of neuronal network. Axons are guided by a variety of guidance factors, such as semaphorins, ephrins and netrin. Plexins function as receptors for the repulsive axonal guidance molecules semaphorins. Intracellular domains of plexins are responsible for initiating cellular signal transduction inducing axon repulsion. Recent advances have revealed molecular mechanisms for plexin-mediated cytoskeletal reorganization, leading to repulsive responses, and small GTPases play important roles in this signaling. Plexin-B1 activates Rho through Rho-specific guanine nucleotide exchange factors, leading to neurite retraction. Plexin-B1 possesses an intrinsic GTPase-activating protein activity for R-Ras and induces growth cone collapse through R-Ras inactivation. In this review we survey current understanding of the signaling mechanisms of plexins.

Semaphorin 4D/Plexin-B1–mediated R-Ras GAP activity inhibits cell migration by regulating β1 integrin activity

Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration (Oinuma, I., Y. Ishikawa, H. Katoh, and M. Negishi. 2004. Science. 305:862–865). We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras–mediated phosphatydylinositol 3-kinase activation, and β1 integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras–specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing β1 integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated β1 integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1–mediated R-Ras GAP activity controls cell migration by modulating the activity of β1 integrins.

Different requirement for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1.

Plexins, comprising Plexin-A, -B, -C, and -D subfamilies, are receptors for semaphorins governing cell adhesion, migration, and axon guidance. Among plexin subfamilies, Plexin-A1 and Plexin-B1 have been shown to function as an R-Ras GAP, inducing repulsive responses, and the expression of R-Ras GAP activity requires the binding of Rnd1, a member of Rnd subfamily of Rho GTPases. However, signaling pathways of Plexin-D1 and Plexin-C1 still remain obscure. Here, we found that Plexin-D1 displayed R-Ras GAP activity and inhibited migration of COS-7 cells, and these actions required Rnd2, another Rnd subfamily GTPase. Rnd2 bound to Plexin-D1 in cortical neurons, and Sema3E/Plexin-D1-induced inhibition of axon outgrowth of cortical neurons required Rnd2 and down-regulation of R-Ras activity. On the other hand, Plexin-C1 displayed R-Ras GAP activity and inhibited cell migration of COS-7 cells without Rnd proteins. Therefore, R-Ras GAP activity is a common function of plexin subfamilies but the regulation of R-Ras GAP activity of plexins by Rnd proteins is different among plexin subfamilies.

Plexin-B1 mutations in prostate cancer

Semaphorins are a large class of secreted or membrane-associated proteins that act as chemotactic cues for cell movement via their transmembrane receptors, plexins. We hypothesized that the function of the semaphorin signaling pathway in the control of cell migration could be harnessed by cancer cells during invasion and metastasis. We now report 13 somatic missense mutations in the cytoplasmic domain of the Plexin-B1 gene. Mutations were found in 89% (8 of 9) of prostate cancer bone metastases, in 41% (7 of 17) of lymph node metastases, and in 46% (41 of 89) of primary cancers. Forty percent of prostate cancers contained the same mutation. Overexpression of the Plexin-B1 protein was found in the majority of primary tumors. The mutations hinder Rac and R-Ras binding and R-RasGAP activity, resulting in an increase in cell motility, invasion, adhesion, and lamellipodia extension. These results identify a key role for Plexin-B1 and the semaphorin signaling pathway it mediates in prostate cancer.

Plexin‐B1 is a GTPase activating protein for M‐Ras, remodelling dendrite morphology

Plexins are receptors for axonal guidance molecules known as semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin‐B1, induces axonal growth cone collapse by functioning as an R‐Ras GTPase activating protein (GAP). Here, we report that Plexin‐B1 shows GAP activity for M‐Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M‐Ras was upregulated during dendritic development. Knockdown of endogenous M‐Ras—but not R‐Ras—reduced dendritic outgrowth and branching, whereas overexpression of constitutively active M‐Ras, M‐Ras(Q71L), enhanced dendritic outgrowth and branching. Sema4D suppressed M‐Ras activity and reduced dendritic outgrowth and branching, but this reduction was blocked by M‐Ras(Q71L). M‐Ras(Q71L) stimulated extracellular signal‐regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppressed ERK activity and down‐regulation of ERK was required for a Sema4D‐induced reduction of dendrite growth. Thus, we conclude that Plexin‐B1 is a dual functional GAP for R‐Ras and M‐Ras, remodelling axon and dendrite morphology, respectively.

R-Ras Controls Axon Specification Upstream of Glycogen Synthase Kinase-3β through Integrin-linked Kinase

The initial event in establishing a polarized neuron is the specification of a single axon. Spatially regulated glycogen synthase kinase-3β (GSK-3β) activity is critical for specifying axon-dendrite fate; however, the upstream signaling of GSK-3β in the determination of neuronal polarity still remains obscure. Here, we found that, in cultured hippocampal neurons, the small GTPase R-Ras selectively localized in a single neurite of stage 2 neurons and that its activity increased after plating and peaked between stages 2 and 3. Ectopic expression of R-Ras induced global inactivation of GSK-3β and formation of multiple axons, whereas knockdown of endogenous R-Ras by RNA interference blocked GSK-3β inactivation and axon formation. GSK-3β inactivation and axon formation by R-Ras required integrin-linked kinase (ILK), and subcellular localization of ILK was strictly regulated by R-Ras-mediated phosphatidylinositol 3-kinase activity. In addition, membrane targeting of ILK was sufficient to inactivate GSK-3β and to form multiple axons. Our study demonstrates a novel role of R-Ras and ILK upstream of GSK-3β in the regulation of neuronal polarity.

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.

R-ras as a key player for signaling pathway of plexins.

Axon guidance represents an important step in the formation of neuronal networks. Axons are guided by various guidance factors, such as semaphorins, slits, ephrins, and netrins. Plexins are cell surface receptors for the repulsive molecules of the semaphorin family. Cytoplasmic regions of plexins are responsible for initiating cellular signal transduction, resulting in axon repulsion. Recent advances have shed light on the signal transduction mechanism of plexins and the mechanisms by which it leads to a repulsive response. Plexin-B1 possesses an intrinsic guanine triphosphate (GTP)ase activating protein activity for R-Ras, a member of Ras family of small GTPases that has been implicated in promoting cell adhesion and neurite outgrowth through integrin activation. Stimulation of Plexin-B1 by Sema4D induces collapse of the growth cone through down-regulation of R-Ras activity. This article summarizes current understanding of the signaling mechanisms of plexins.