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

Abstract

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.

Figure 1

Sema4D suppresses R-Ras activity, dephosphorylates Akt and GSK-3β and phosphorylates CRMP2 in cultured hippocampal neurons. Neurons dissociated from rat embryos at embryonic day 18.5 were stimulated at 3 days in vitro (d.i.v.) with Sema4D for 1.5 h. (A) Activity of R-Ras with or without Sema4D stimulation. GTP-bound R-Ras isolated with GST–RBD was detected with an anti-R-Ras antibody. Relative activity of R-Ras was determined by the amount of R-Ras bound to GST–RBD normalized to the amount of R-Ras in cell lysates analysed by National Institutes of Health Image software. (B–D) Analysis of phosphorylated (p)-Akt, p-GSK-3β and p-CRMP2. Cell lysates were analysed by immunoblot analysis with the phospho-specific antibodies against Akt (Ser 473; B), GSK-3β (Ser 9; C) and CRMP2 (Thr 514; D). Results are the means±s.e.m. of three independent experiments. CRMP2, collapsin response mediator protein 2; GSK-3β, glycogen synthase kinase-3β; GST–RBD, glutathione S-transferase-fused Ras-binding domain; Sema4D, semaphorin 4D.

Figure 2

Constitutively active mutants of R-Ras, PI(3)K, Akt, and GSK-3 inhibitors suppress the Sema4D-induced growth cone collapse. (A) Inhibition of Sema4D-induced growth cone collapse by constitutive activation of R-Ras, PI(3)K and Akt. Neurons at 2 days in vitro (d.i.v.) were transfected with the indicated plasmids, and were fixed at 3 d.i.v. after stimulation with Sema4D for 1.5 h. F-actin was stained with rhodamine-conjugated phalloidin to visualize the lamellipodia and filopodia. (B) Inhibition of Sema4D-induced growth cone collapse by GSK-3 inhibitors. Neurons pretreated with inhibitors for GSK-3, SB-216763 and LiCl were used in the collapse assay. The results are the means±s.e.m. of three independent experiments in which at least 20 cells were counted. Scale bar, 10 μm. DMSO, dimethyl sulphoxide; GFP, green fluorescent protein; GSK-3, glycogen synthase kinase-3; PI(3)K, phosphatidylinositol-3-OH kinase; Sema4D, semaphorin 4D.

Figure 3

Sema4D dephosphorylates Akt and GSK-3β and phosphorylates CRMP2 through the conserved R-Ras GAP domain within plexin-B1. (A) Time course of Sema4D-induced dephosphorylation of Akt and GSK-3β. COS-7 cells expressing full-length plexin-B1 and Rnd1 were stimulated with Sema4D for the indicated times. The cell lysates at the indicated times were analysed by immunoblot analysis with the phospho-specific antibodies against Akt and GSK-3β. (B) Requirement of Rnd1 in Sema4D-induced dephosphorylation of Akt and GSK-3β. COS-7 cells expressing the indicated plasmids were stimulated with Sema4D for 3 min, and the cell lysates were analysed by immunoblot analysis with phospho-specific antibodies against Akt and GSK-3β. (C–F) Effects of plexin-B1Δect mutants on phosphorylation of Akt, GSK-3β and CRMP2. Lysates from COS-7 cells expressing the indicated plasmids were analysed by immunoblot analysis with phospho-specific antibodies against Akt, GSK-3β and CRMP2. (G) Plexin-A1-mediated dephosphorylation of Akt and GSK-3β. Lysates from COS-7 cells expressing plexin-A1Δect mutants and Rnd1 were analysed by immunoblot analysis with phospho-specific antibodies against Akt and GSK-3β. (H) Dephosphorylation of Akt and GSK-3β by suppression of R-Ras activity. Lysates from COS-7 cells expressing myr-R-RasGAP were analysed by immunoblot analysis with phospho-specific antibodies against Akt and GSK-3β. The results shown are representative of two or three experiments. CRMP2, collapsin response mediator protein 2; GAP, GTPase-activating protein; GFP, green fluorescent protein; GSK-3β, glycogen synthase kinase-3β; HA, haemagglutinin; Sema4D, semaphorin 4D; WT, wild type.

Figure 4

Inhibition of R-Ras-mediated PI(3)K activation or constitutive activation of GSK-3β is sufficient to induce growth cone collapse. Neurons were transfected with the indicated plasmids at 2 days in vitro (d.i.v.) and neurons at 3 days in vitro (d.i.v.) were fixed. Alternatively, cells were treated with LY294002 for 1.5 h before fixation. F-actin was stained with rhodamine-conjugated phalloidin to visualize the lamellipodia and filopodia. The results are the means±s.e.m. of three independent experiments in which at least 20 cells were counted. Scale bar, 10 μm. GAP, GTPase-activating protein; GFP, green fluorescent protein; GSK-3β, glycogen synthase kinase-3β; KD, kinase dead.