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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2010 September 3; 285(36): 28200–28209.
Published online 2010 July 7. doi:  10.1074/jbc.M110.147546
PMCID: PMC2934685

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

Abstract

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.

Keywords: Axon, Neuron, Phosphatidylinositol 3-Kinase, Ras, Semaphorin

Introduction

Semaphorins (Sema)2 are a large family of secreted or membrane-bound molecules that play central roles in axon guidance in developing nervous system (1,3). In addition to the nervous system, they are widely expressed in embryonic and adult tissues and mediate diverse biological processes, such as cardiac and skeletal development (4), tumor growth, and metastasis (5), and the immune response (6). The functions of semaphorins are mediated by plexins, single-pass transmembrane receptors, which are classified into four subfamilies: Plexin-A1 to -4, Plexin-B1 to -3, Plexin-C1, and Plexin-D1 (2, 7). Plexin-B1 has been identified as a receptor for semaphorin 4D (Sema4D) (2). Semaphorins were originally identified as repulsive and attractive axonal guidance molecules, and Sema4D/Plexin-B1 induces axonal growth cone collapse in hippocampal neurons (8, 9). Rho family small GTPases are signal transduction molecules that remodel the actin cytoskeleton and play fundamental roles in numerous cellular processes (10, 11). First, Plexin-B1 has been shown to activate RhoA through the interaction of its intracellular COOH-terminal PSD-95/Dlg/ZO-1 (PDZ) domain-binding motif with PDZ-Rho guanine nucleotide exchange factor (GEF)/leukemia-associated RhoGEF (LARG) and induce neurite retraction through the RhoA/ROCK signaling pathway (8, 13, 14). However, the PDZ domain-binding motif is restricted to the Plexin-B subfamily. Conversely, the cytoplasmic regions are highly conserved among all plexin subfamilies from Caenorhabditis elegans to humans. We previously reported that the small GTPase Rnd1, a constitutively active Rho family GTPase, interacts directly with the cytoplasmic domain of Plexin-B1 (15). We further revealed that the cytoplasmic region of Plexin-B1 directly encodes a GTPase-activating protein (GAP) for R-Ras, and Plexin-B1 specifically down-regulates R-Ras activity in response to Sema4D, inducing axonal growth cone collapse in hippocampal neurons, and that the expression of R-Ras GAP activity of Plexin-B1 requires Rnd1 association with the receptor (16). Furthermore, it has been shown that other plexin subfamilies, including Plexin-A, -C, and -D, also display R-Ras GAP activity, and this activity is required for semaphorin-induced repulsive response, indicating that R-Ras GAP activity is a common signaling activity of the plexin family (17, 18). In addition, we recently revealed that Plexin-B1 displays a GAP activity for M-Ras, another member of R-Ras subfamily GTPase, remodeling dendrite morphology in cortical neurons (19).

We characterized the downstream signaling of Plexin-B1-mediated R-Ras GAP activity for repulsive response, and we found that Plexin-B1 suppresses the PI3K signaling pathway and dephosphorylates Akt and GSK-3β (glycogen synthase kinase-3β) through R-Ras GAP activity, inducing growth cone collapse (20). R-Ras is known to preferentially activate PI3K, producing phosphatidylinositol 3,4,5-trisphosphate (PIP3) (21, 22). PIP3 is a key molecule in a variety of biological functions, including neuronal migration and axon elongation, through modulation of a variety of signaling molecules, such as Akt, integrin-linked kinases, phosphoinositide-dependent kinases, and GEFs (23). Levels of PIP3 are critically based on the balance between activity of PI3K and PTEN (phosphatase and tensin homologue deleted on chromosome ten), which is a PI3-phosphatase, decreasing the level of PIP3 (24). PTEN is a critical player of the PI3K signaling pathway and regulates many physiologically and pathologically significant processes, such as cellular proliferation, survival, growth, and motility (24). PTEN is known to be a tumor suppressor, which antagonizes motility, inhibits cell cycle progression, and induces apoptosis in a variety of cells through the inhibition of PI3K signaling, and loss of PTEN function causes tumorigenesis or abnormality in neural development (24). The PI3-phosphatase activity of PTEN is critically regulated by phosphorylation of a cluster of serine and threonine residues located in the COOH-terminal region (25). Phosphorylation of the COOH-terminal region keeps PTEN as a closed inactive conformation, and dephosphorylation renders PTEN open and active in conformation and allows it to associate with the plasma membranes, displaying phosphatase activity (26). It has been reported that alanine substitutions at these phosphorylation sites of PTEN (PTEN-3A; a phosphorylation-resistant mutant) lead to higher catalytic activity, whereas the mutant with glutamic acid substitutions at these sites (PTEN-3E; a phosphorylation mimic mutant) shows low activity, acting as a dominant negative form (27). Thus, the dephosphorylation is a key step for the activation of PTEN. There are several protein kinases that potentially phosphorylate the COOH-terminal domain of PTEN, and among them CK2 (casein kinase 2) has been implicated in the phosphorylation and inhibition of PTEN activity (28). CK2 is a serine/threonine protein kinase that consists of a catalytic α subunit and regulatory β subunit (29). CK2 has a variety of physiological targets and participates in a series of cellular functions, including the maintenance of cell viability (29). It was recently reported that nerve growth factor, a member of axon-growing neurotrophins, activates CK2 and then phosphorylates and suppresses PTEN activity, promoting axon outgrowth, suggesting that CK2-mdiated PTEN inhibition is involved in the regulation of axon outgrowth (30).

Concerning a role of PTEN in axon guidance, the involvement of PTEN in the Sema3A-mediated growth cone collapse has been suggested (31). However, signaling pathways of semaphorin/plexins, including PTEN, remain elusive. In this study, we characterized the downstream signaling pathway of Sema4D/Plexin-B1-mediated R-Ras GAP activity for growth cone collapse and showed that Sema4D/Plexin-B1 inhibits CK2α activity and promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse in hippocampal neurons.

EXPERIMENTAL PROCEDURES

Plasmid Constructs

Hemagglutinin (HA)-tagged Rnd1, green fluorescent protein (GFP)-tagged human R-Ras and R-Ras-QL (Q87L), the NH2-terminal HA-tagged myristoylated form of the R-Ras GAP domain of R-Ras GAP (amino acids 291–614), Myc-tagged full-length human Plexin-B1, Myc-Plexin-B1-ΔC (lacking the last seven COOH-terminal amino acids), Myc-Plexin-B1-Δect (deletion of amino acids 1–1306), Myc-Plexin-B1-Δect-RA (R1677A/R1678A/R1984A), and Myc-Plexin-B1-Δect-GGA (L1849G/V1850G/P1851A), have been described previously (16, 32, 33). cDNAs encoding rat PTEN and CK2α were obtained by PCR from adult rat brains and subcloned into pEGFP (Clontech). GFP-tagged PTEN mutants, GFP-PTEN-C124S (a phosphatase activity-deficient dominant negative mutant), GFP-PTEN-3A (S380A/T382A/T383A) (a phosphorylation-resistant mutant), and GFP-PTEN-3E (S380E/T382E/T383E) (a phosphorylation-mimic mutant), and a CK2α mutant, CK2α–K68A (a mutant lacking kinase activity), were generated by PCR-mediated mutagenesis. The short hairpin RNAs (shRNAs) for PTEN were designed to target 19 nucleotides of rat transcripts and expressed by using an shRNA expression vector, pSilencer (Ambion). The target sequences for PTEN shRNA constructs are as follows: PTEN shRNA 338 (nucleotides 338–356, 5′-GTGAAGACGACAATCATGT-3′) and PTEN shRNA 1064 (nucleotides 1064–1084, 5′-CAAATCCAGAGGCTAGCAGTT-3′).

Antibodies and Reagents

Antibodies used were as follows: mouse monoclonal antibodies against Myc and Na+/K+-ATPase α subunit (Upstate Biotechnology); mouse monoclonal antibodies against α-tubulin and vimentin (Sigma); mouse monoclonal antibody against GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); rabbit polyclonal antibodies against Akt phosphorylated at Ser-473, Akt, PTEN phosphorylated at Ser-380, and PTEN (Cell Signaling); a rat monoclonal antibody against HA (Roche Applied Science); a mouse monoclonal antibody against GAPDH (Ambion); horseradish peroxidase-conjugated secondary antibodies (DAKO); Alexa 594-conjugated secondary antibodies and rhodamine-phalloidin (Molecular Probes). The pharmacological PTEN inhibitor dipotassium bisperoxo (picolinato) oxovanadate (V) (bpV(pic)) was purchased from Calbiochem and dissolved in Me2SO. Laminin and poly-l-lysine were purchased from Sigma and used as 10 μg and 25 μg/ml, respectively. A soluble form of Sema4D fused to human IgG1-Fc was from H. Kikutani (Osaka University, Osaka, Japan), and Sema4D stimulation was carried out by replacing the culture medium with a Sema4D-containing conditioned medium, which contained ~150 nm Sema4D-Fc.

Cell Culture and Transfection

COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mm glutamine, 100 units/ml penicillin, and 0.2 mg/ml streptomycin under humidified conditions in 95% air and 5% CO2 at 37 °C. Transient transfections were carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and the cultures were maintained in DMEM with 0.5% FBS after transfection for 2.5 days. Primary hippocampal neurons were isolated from embryonic day 18.5 rats as described previously (20) and were plated onto the poly-l-lysine- and laminin-coated coverslips (circular, 13 mm in diameter) or plastic dishes (60 mm in diameter), at a density of 3.5 × 104 cells/cm2. After 2 days in culture, the medium was changed to Opti-MEM (Invitrogen) supplemented with 2% B-27 (Invitrogen), and the neurons were transfected with test plasmids using Lipofectamine 2000 for analysis of growth cone morphology. Neurons were fixed with 4% paraformaldehyde and in phosphate-buffered saline (PBS) and processed for the immunohistochemistry. Hippocampal neurons dissociated from embryonic day 18.5 rats were transfected with test plasmids using the Rat Neuron Nucleofector kit (Amaxa Biosystems) following the manufacturer's instructions for immunoblotting.

Measurement of Growth Cone Collapse

Immunocytochemistry was performed as described previously (16). Briefly, all steps were carried out at room temperature, and neurons were rinsed with PBS between each step. After transfection, neurons on coverslips were fixed with 4% formaldehyde in PBS for 15 min. After residual formaldehyde had been quenched with 50 mm NH4Cl in PBS for 10 min, neurons were permeabilized with 0.2% Triton X-100 in PBS for 10 min and incubated with 10% FBS in PBS for 30 min to block nonspecific antibody binding. To visualize filamentous actin (F-actin), fixed neurons were stained with rhodamine-phalloidin. Neurons on coverslips were mounted in 90% glycerol containing 0.1% p-phenylenediamine dihydrochloride in PBS. Neuronal morphology was analyzed from digital images acquired at ×40 magnification by using a Leica DC350F digital camera system equipped with a Nikon Eclipse E800 microscope and Image-Pro Plus image analysis software (Media Cybernetics). Tips of the longest neurites were analyzed, and growth cone positive cells were defined as those that possessed lamellipodia and filopodia. More than 30 cells were examined in three independent experiments, and statistical significance was determined by using the analysis of variance test (Dunnett) or Student's t test.

Preparation of Cytoskeletal Membrane Fractions

Preparation of Triton X-100-soluble and -insoluble fractions of neurons or COS-7 cells was performed as described elsewhere (34). Cells were washed with cell solubilization buffer (10 mm PIPES, pH 7.0, 50 mm KCl, 10 mm EGTA, 3 mm MgCl2, 2 m glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 mm NaF, and 1 mm NaVO3) and then lysed with the buffer, containing 1% Triton X-100, for 1 min at 4 °C by homogenizing with a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 100,000 × g for 1 h to remove the soluble fraction. The particulate pellet was resuspended in extraction buffer (20 mm Tris-HCl, pH 7.5, 80 mm KCl, 1 mm EGTA, 30 mm MgCl2, 0.25 m NaCl, 10 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin), and the fraction was passed through a 25-gauge syringe three times before immunoblotting analysis.

Immunoblotting

Proteins were separated by 7 or 12.5% SDS-PAGE and were electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore Corp.). The membrane was blocked with 3% low fat milk in Tris-buffered saline (10 mm Tris-HCl, pH 7.5, 150 mm NaCl) and then incubated with primary antibodies. The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and a chemiluminescence detection kit (Chemi-Lumi One, Nacalai Tesque).

Measurement of PTEN Phosphatase Activity

Measurement of PTEN phosphatase activity in cells was performed as described previously (35). Neurons or COS-7 cells were lysed with ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm NaVO3, 25 mm NaF, 100 μm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 mm dithiothreitol). After centrifugation at 100,000 × g for 1 h, the extracts were incubated with anti-PTEN antibody for 2 h at 4 °C and then with Protein G-Sepharose beads (GE Healthcare) for 1 h. The PTEN phosphatase enzyme reaction was initiated by the incubation of immunoprecipitates in the reaction buffer containing 1 nmol of PIP3 (WAKO) and 10 nmol of phosphatidylserine (Sigma) for 30 min at 25 °C. The reaction was terminated by the addition of N-methylimide. After centrifugation, the supernatant was incubated with Biomol Green (Biomol) for 20 min at 25 °C, and the color was quantified by spectrophotometry at 620 nm, which reflected the relative amount of PTEN activity in the sample.

Measurement of CK2α Kinase Activity

CK2α kinase activity was measured using a CK2 kinase assay kit (CycLex). Neurons were harvested and sonicated in Kinase Reaction Buffer. After centrifugation, the extracts were pipetted into the 96-well plates precoated with a substrate of CK2α corresponding to recombinant p53 (amino acids 1–55), and incubated for 10 min at 30 °C in the presence of 12.5 μm ATP. After the reaction of samples containing CK2α and precoated recombinant p53, the wells were washed with buffer five times and incubated with the horseradish peroxidase-conjugated anti-phospho-p53 (Ser-46) antibody (TK-4D4) for 30 min at 25 °C. After the incubation with TK-4D4, the wells were washed with wash buffer five times and incubated with the TMB for 5 min at 25 °C. After the peroxidase color reaction, the reaction was terminated by the addition of H2SO4, and the color was quantified by spectrophotometry at 450 nm, which reflected the relative amount of CK2 activity in the sample.

RESULTS

Sema4D Stimulates PTEN Activity

It has been shown that dephosphorylation of PTEN at the COOH-terminal region allows PTEN to associate with the membrane, leading to PTEN activation, indicating that the dephosphorylation and subsequent membrane translocation of PTEN are critical steps for PTEN activation (25). We examined the effect of Sema4D on the phosphorylation level of PTEN at Ser-380 in primary cultured rat hippocampal neurons. As shown in Fig. 1A, Sema4D stimulation caused a marked decrease in phosphorylated PTEN at Ser-380. To study the effect of Sema4D on subcellular localization of PTEN, we prepared a Triton X-100-soluble fraction and insoluble cytoskeletal membrane fraction from cultured hippocampal neurons. Sema4D stimulation induced the increase in the amount of PTEN protein in the Triton X-100-insoluble membrane fraction in parallel with the decrease in the soluble fraction (Fig. 1B). We further examined the effect of Sema4D on the in vitro phosphatase activity of PTEN. Sema4D significantly stimulated the PTEN phosphatase activity in hippocampal neurons (Fig. 1C). Thus, Sema4D stimulated dephosphorylation of PTEN, the shift in its Triton X-100 extractability, and in vitro PTEN phosphatase activity.

FIGURE 1.
Sema4D stimulates PTEN activity. Neurons dissociated from embryonic day 18.5 rats were stimulated at 3 days in vitro (DIV) with or without Sema4D for 1.5 h. A, cell lysates were analyzed by immunoblotting with antibodies against phospho-PTEN (p-PTEN) ...

PTEN Activity Is Required for Sema4D-induced Growth Cone Collapse

To assess the role of PTEN in Sema4D-induced growth cone collapse, we generated two shRNA expression vectors designed to target two different regions of the PTEN transcript; these shRNAs (338 and 1064) effectively reduced the amount of endogenous PTEN in hippocampal neurons (see Fig. 3A). Hippocampal neurons were transfected with GFP and shRNAs, and their growth cone morphology was observed. In neurons expressing the control shRNA, Sema4D normally induced growth cone collapse, but the expression of two PTEN-specific shRNAs (338 and 1064) impaired the Sema4D-induced growth cone collapse (Fig. 2A). We next examined the effect of PTEN-C124S, a phosphatase activity-deficient dominant negative mutant of PTEN, on the Sema4D-induced growth cone collapse. The expression of PTEN-C124S strongly suppressed the Sema4D-induced growth cone collapse (Fig. 2B). We further examined the effect of bpV(pic), a pharmacological PTEN inhibitor, on the Sema4D-induced growth cone collapse. Pretreatment with bpV(pic) also suppressed the Sema4D-induced growth cone collapse (Fig. 2C). In contrast, the expression of wild-type PTEN by itself triggered the growth cone collapse (Fig. 2D). Previously, we demonstrated that Sema4D induces dephosphorylation of Akt, and this Akt dephosphorylation is a critical step for inducing growth cone collapse in hippocampal neurons (20). We then examined the effects of two PTEN-specific shRNAs on the Sema4D-inducd Akt dephosphorylation in hoppocampal neurons. Expression of two PTEN shRNAs (338 and 1064) but not control shRNA inhibited the Sema4D-induced dephosphorylation of Akt (Fig. 3B). These results indicate that PTEN activity is required for the Sema4D-induced Akt dephosphorylation and subsequent growth cone collapse.

FIGURE 2.
PTEN is required for Sema4D-induced growth cone collapse. A, neurons at 2 DIV were transfected with control shRNA or PTEN shRNA (338 or 1064) and were fixed at 3 DIV after stimulation with or without Sema4D for 1.5 h. B, neurons at 2 DIV were transfected ...
FIGURE 3.
PTEN is required for Sema4D-induced dephosphorylation of Akt. A, neurons were transfected with control shRNA or PTEN shRNA (338 or 1064), before plating, using nucleofection technology, and cell lysates at 3 DIV were analyzed by immunoblotting with antibodies ...

Dephosphorylation of PTEN Is Required for Sema4D-induced Growth Cone Collapse

Dephosphorylation of PTEN is a critical step for PTEN activation (24). To evaluate the role of Sema4D-induced dephosphorylation of PTEN in the growth cone collapse, we constructed two GFP-tagged PTEN mutants, GFP-PTEN-3A (S380A/T382A/T383A), a phosphorylation-resistant mutant, and GFP-PTEN-3E (S380E/T382E/T383E), a phosphorylation-mimic mutant. In COS-7 cells, expression of GFP-PTEN-3A induced dephosphorylation of Akt, whereas GFP-PTEN-3E expression significantly promoted the Akt phosphorylation, suggesting that PTEN-3A and PTEN-3E act as constitutively active and dominant negative mutants, respectively (Fig. 4A). We first examined the effect of GFP-PTEN-3E, the dominant negative mutant, on the Sema4D-induced growth cone collapse in hippocampal neurons. As shown in Fig. 4B, the expression of GFP-PTEN-3E strongly suppressed the Sema4D-induced growth cone collapse (Fig. 4B). We next examined the effect of GFP-PTEN-3A, the constitutively active mutant, on the growth cone morphology. The expression of GFP-PTEN-3A by itself triggered growth cone collapse (Fig. 4C). These results strongly indicate that the Sema4D-induced dephosphorylation of PTEN is necessary for the Sema4D-induced growth cone collapse in hippocampal neurons.

FIGURE 4.
Effects of phosphorylation-resistant and phosphorylation-mimic PTEN mutants on Akt phosphorylation and growth cone collapse. A, COS-7 cells were transfected with GFP-PTEN-3A or GFP-PTEN-3E, and cell lysates were analyzed by immunoblotting with antibodies ...

Sema4D/Plexin-B1 Induces Dephosphorylation of PTEN through R-Ras GAP Activity

We previously demonstrated that Sema4D/Plexin-B1 induces the growth cone collapse through R-Ras GAP activity and that the expression of R-Ras GAP activity requires the binding of Rnd1, a member of the Rho family and a constitutively active GTPase, to the intracellular cytoplasmic domain of Plexin-B1 (16). We then examined whether the Sema4D-induced PTEN dephosphorylation is mediated by R-Ras GAP activity of Plexin-B1 in COS-7 cells ectopically expressing Plexin-B1 and Rnd1. As shown in Fig. 5A, Sema4D stimulation markedly induced dephosphorylation of PTEN at Ser-380 in COS-7 cells expressing wild-type full-length Plexin-B1 and Rnd1. It has been reported that Plexin-B1 stimulates RhoA activity through the interaction of its COOH-terminal PDZ domain-binding motif with PDZ-RhoGEF/LARG (8, 13, 14). To study whether Plexin-B1-induced RhoA activation is involved in the PTEN dephosphorylation, we examined the PTEN phosphorylation in COS-7 cells expressing Plexin-B1-ΔC, lacking the COOH-terminal PDZ domain-binding motif, which has no ability to stimulate RhoA activity (33). Sema4D stimulation markedly induced dephosphorylation of PTEN in COS-7 cells expressing Plexin-B1-ΔC and Rnd1, the decreased level of phosphorylated PTEN being the similar to that of wild-type full-length Plexin-B1 (Fig. 5B). Thus, the Sema4D/Plexin-B1-induced PTEN dephosphorylation is not mediated by RhoA activation. We recently reported that the extracellular domain-deleted Plexin-B1, Plexin-B1Δect, shows constitutive, ligand-independent activity of R-Ras GAP (33). We next examined the effect of two mutants of Plexin-B1Δect: Plexin-B1Δect-RA, lacking catalytic activity of R-Ras GAP, and Plexin-B1Δect-GGA, lacking Rnd1-interacting activity, on PTEN phosphorylation. In COS-7 cells, Plexin-B1Δect induced the decrease in PTEN phosphorylation level in the presence of Rnd1 but not in the absence of Rnd1 (Fig. 5C). Two mutants, Plexin-B1Δect-RA and Plexin-B1Δect-GGA, failed to induce the decrease in PTEN phosphorylation level. In addition, expression of R-Ras(Q87L), a constitutively active mutant of R-Ras, antagonized the Plexin-B1Δect/Rnd1-mediated decrease in PTEN phosphorylation (Fig. 5D). These results indicate that Plexin-B1 dephosphorylates PTEN through R-Ras GAP activity but not RhoA activation.

FIGURE 5.
Sema4D induces dephosphorylation of PTEN through R-Ras GAP activity. A, COS-7 cells were transfected with Myc-tagged full-length Plexin-B1 and HA-tagged Rnd1, and they were stimulated with Sema4D for the indicated times. The cell lysates at the indicated ...

To next study whether R-Ras GAP activity is sufficient for the activation of PTEN, the myristoylated GAP domain of R-Ras GAP, which shows specific GAP activity toward R-Ras (36), was transfected, and the phosphorylation level of PTEN at Ser-380, subcellular localization of PTEN, and in vitro PTEN phosphatase activity were examined. The expression of the myristoylated GAP domain of R-Ras GAP induced the decrease in phosphorylation level of PTEN in hippocampal neurons (Fig. 6A), the increase in the amount of PTEN in the Triton X-100-insoluble membrane fraction in parallel with the decrease in the soluble fraction (Fig. 6B), and stimulation of in vitro PTEN phosphatase activity in COS-7 cells (Fig. 6C). These results indicate that R-Ras GAP activity is sufficient for dephosphorylation and subsequent activation of PTEN.

FIGURE 6.
R-Ras GAP activity stimulates PTEN activity. A, neurons at 2 DIV were transfected with HA-tagged myr-R-Ras GAP domain of R-Ras GAP before plating, using nucleofection technology. Cell lysates at 3 DIV were analyzed by immunoblotting with antibodies against ...

CK2α Is Involved in Sema4D-induced PTEN Dephosphorylation and Growth Cone Collapse

Sema4D/Plexin-B1 induces dephosphorylation of PTEN through R-Ras GAP activity. We next tried to reveal a signaling pathway of Sema4D/Plexin-B1 for R-Ras GAP-mediated PTEN dephosphorylation, leading to growth cone collapse. CK2α is one of the potential protein kinases for PTEN phosphorylation, regulating PTEN activity (28). We then studied whether Sema4D/Plexin-B1 regulates CK2α activity. We first examined the effect of Sema4D on in vitro CK2α kinase activity in hippocampal neurons. Sema4D stimulation induced the significant decrease in in vitro CK2α kinase activity (Fig. 7A). To study the involvement of CK2α in the Sema4D-induced growth cone collapse, hippocampal neurons were transfected with GFP-CK2α WT. The overexpression of GFP-CK2α WT strongly suppressed the Sema4D-induced growth cone collapse, suggesting that down-regulation of CK2α activity is required for the Sema4D-induced growth cone collapse (Fig. 7B). We next studied the role of CK2α in Plexin-B1-induced PTEN dephosphorylation in COS-7 cells. The overexpression of wild-type CK2α blocked the Plexin-B1Δect/Rnd1-induced dephosphorylation of PTEN at Ser-380 in COS-7 cells (Fig. 7C). On the other hand, the expression of R-Ras-QL stimulated the phosphorylation of PTEN, but this stimulation was strongly suppressed by the expression of CK2α-K68A, a mutant lacking kinase activity (Fig. 7D). Furthermore, expression of CK2α-K68A significantly induced growth cone collapse in hippocampal neurons (Fig. 7E). We further examined the effect of myristoylated R-Ras GAP domain of R-Ras GAP on in vitro kinase activity of CK2α. The expression of the R-Ras GAP domain strongly suppressed the kinase activity of CK2α in COS-7 cells (Fig. 7F). These results suggest that R-Ras stimulates PTEN phosphorylation through CK2α and that the R-Ras GAP activity of Plexin-B1 induces PTEN dephosphorylation through the inhibition of CK2α activity.

FIGURE 7.
CK2α is involved in Sema4D-induced PTEN dephosphorylation and growth cone collapse. A, neurons at 3 DIV were stimulated with or without Sema4D for 1.5 h. CK2α kinase activity in cell lysates was determined as described under “Experimental ...

DISCUSSION

We previously reported that Sema4D receptor Plexin-B1 induces growth cone collapse by functioning as a GAP for R-Ras, a member of the Ras family GTPase implicated in axon outgrowth (16). In this study, we observed the downstream signaling of Plexin-B1-mediated R-Ras GAP activity and showed that Sema4D/Plexin-B1 induces dephosphorylation and activation of PTEN downstream of R-Ras GAP activity, inducing growth cone collapse in hippocampal neurons.

Recently, it was reported that active RhoA induces membrane translocation and activation of PTEN through its phosphorylation by ROCK, a downstream RhoA effector (37). Plexin-B1 is known to activate RhoA through direct association with PDZ-RhoGEF/LARG by means of its COOH-terminal PDZ-domain-binding motif, and this activation is in part involved in the repulsive functions of Plexin-B1 (8, 13, 14). However, a Plexin-B1 mutant, Plexin-B1-ΔC, lacking the COOH-terminal PDZ-domain-binding motif, induces dephosphorylation of PTEN, indicating that the RhoA activation signal of Plexin-B1 is not involved in dephosphorylation of PTEN. Conversely, Plexin-B1-RA, lacking the catalytic activity of R-Ras GAP, fails to induce dephosphorylation of PTEN. In addition, myr-R-Ras GAP domain of R-Ras GAP by itself induces dephosphorylation of PTEN. Thus, Plexin- B1 induces dephosphorylation of PTEN through R-Ras GAP activity, and R-Ras GAP activity is enough for induction of PTEN dephosphorylation.

Concerning downstream signaling of plexins, we recently demonstrated that Sema4D/Plexin-B1 dephosphorylates Akt and GSK-3β and promotes phosphorylation of CRMP-2 (collapsing response mediator protein-2) through R-Ras GAP activity, inducing growth cone collapse (20). Sema3A has been also shown to induce dephosphorylation of Akt and GSK-3β and promotes phosphorylation of CRMP-2, leading to growth cone collapse (38, 39). Akt is a key protein kinase in the PI3K signaling pathway and is phosphorylated and activated in response to the elevation of PIP3 level (40). GSK-3β possesses a high basal kinase activity, and its kinase activity is inhibited through the phosphorylation at Ser-9 by activated Akt (41). The downstream targets of GSK-3β in axon are CRMP-2 and APC (adenomatous polyposis coli), both of which are microtubule-binding proteins that promote microtubule polymerization and stabilization (42). Phosphorylation of CRMP-2 and APC by GSK-3β suppresses their abilities to bind to microtubules, leading to axon repulsion. Thus, inactivation of Akt leads to dephosphorylation and activation of GSK-3β and resultant phosphorylation and inactivation of CRMP-2 and APC, causing microtubule destabilization and growth cone collapse (43, 44). In this signaling pathway, the regulation of PIP3 level is an important step. The level of PIP3 is regulated by both PI3K and PTEN. In this work, we demonstrated that Sema4D/Plexin-B1 activates PTEN and that PTEN activity is required for the Sema4D-mediated Akt dephosphorylation and growth cone collapse in hippocampal neurons. The involvement of PTEN in Sema3A-mediated Akt dephosphorylation and growth cone collapse was also reported (31). PTEN is likely to be a critical regulator in semaphorin-mediated repulsive response. On the other hand, PI3K is known to be the predominant effector for R-Ras, and R-Ras activation leads to PI3K activation and Akt phosphorylation (21, 22). Therefore, we speculate that inhibition of PI3K activation by R-Ras GAP activity and concurrent activation of PTEN act together to induce Sema4D-mediated growth cone collapse.

Meanwhile, Plexin-B1 directly interacts with PDZ-RhoGEF/LARG via its COOH-terminal PDZ domain-binding motif to induce RhoA activation and growth cone collapse in response to Sema4D (8, 13, 14). Previous work, including our studies, demonstrated that Sema4D/Plexin-B1-induced growth cone collapse requires both R-Ras GAP activity and the PDZ-RhoGEF/LARG-mediated RhoA activation (45). As mentioned above, R-Ras GAP activity modulates Akt-GSK-3β-CRMP-2 signaling, regulating microtubule reorganization. On the other hand, RhoA activates ROCK, leading to actomyosin contraction and subsequent axon retraction (45). Thus, R-Ras GAP activity and the PDZ-RhoGEF/LARG-mediated RhoA activation may in concert participate in the Sema4D/Plexin-B1-induced growth cone collapse through their distinct signaling systems, R-Ras GAP/Akt/GSK-3β/CRMP-2-mediated microtubule reorganization and RhoA/ROCK-mediated actomyosin contraction.

The activity of PTEN is regulated by phosphorylation at the cluster of serine/threonine residues, including Ser-380, Thr-382, and Thr-383, located in the COOH-terminal tail domain (25). The phosphorylated COOH-terminal tail intramolecularly interacts with the NH2-terminal core region, including the C2 domain, which has been implicated in plasma membrane binding of PTEN (26). This interaction keeps PTEN closed in conformation and hinders the membrane binding of PTEN. Dephosphorylation of the COOH-terminal region renders PTEN open and active in conformation and allows it to associate with the plasma membranes, displaying phosphatase activity (26). We revealed here that Sema4D/Plexin-B1 induces dephosphorylation of PTEN and stimulates in vitro PTEN activity. In addition, we showed that PTEN-3E, the phosphorylation mimic mutant, inhibits the Sema4D-mediated growth cone collapse, whereas PTEN-3A, the phosphorylation-resistant mutant, by itself induces growth cone collapse. Therefore, Sema4D-incuced dephosphorylation of PTEN is the essential step for the Sema4D-mediated growth cone collapse, and then the dephosphorylation is enough for the induction of growth cone collapse. Recently, PTEN has been reported to directly interact with myosin V dependent on phosphorylation by GSK-3β, leading to the myosin V-mediated translocation of PTEN to plasma membranes (46). Considering that GSK-3β-mediated phosphorylation of PTEN is critical for this translocation, this system cannot account for Sema4D/Plexin-B1-induced membrane translocation and activation of PTEN. PTEN has been shown to associate with a variety of signaling molecules in part a COOH-terminal tail phosphorylation-dependent manner (26). PTEN membrane localization may be mediated by distinct regulatory mechanisms within different cellular contexts.

Sema4D-induced growth cone collapse is an acute response, and longer exposure to Sema4D induces a variety of responses, such as axon branch elimination and axon pruning in neurons through various signaling systems (47). Concerning PTEN activity, we observed that longer exposure to Sema4D induced prolonged PTEN activation, leading to down-regulation of PTEN protein in neurons (data not shown). It has been reported that the phosphorylated inactive form of PTEN is stable, but dephosphorylated active PTEN protein becomes unstable, undergoing proteosomal degradation (28). Sema4D-induced PTEN activation and growth cone collapse appear to be an acute response.

Sphingosine 1-phosphate, a bioactive lipid, is known to be a repulsive factor, and sphingosine 1-phosphate stimulation of its GPCR has been reported to induce the interaction of the receptor and PTEN and PTEN activation, and this PTEN activation is required for the receptor signaling (48). The Sema3A-mediated growth cone collapse has been suggested to utilize PTEN as a signaling intermediate (31). We here demonstrated that Sema4D/Plexin-B1 stimulates PTEN activity through R-Ras GAP activity, inducing growth cone collapse. PTEN may be implicated in signaling systems of several axonal repulsive response factors.

We further examined the signaling pathway for Plexin-B1-induced dephosphorylation of PTEN. PTEN has been shown to be phosphorylated by a series of protein kinases, including CK2 (12). We showed that Sema4D suppresses the activity of CK2α and that overexpression of CK2α abrogates the Sema4D-induced PTEN dephosphorylation and growth cone collapse, suggesting that these actions of Sema4D/Plexin-B1 are mediated by the inhibition of CK2α activity. Furthermore, the expression of CK2α-K68A, the mutant lacking kinase activity, induces growth cone collapse. Thus, inactivation of CK2α is sufficient for the growth cone collapse. Nerve growth factor is a well known neurotrophin, promoting axon outgrowth, and it was recently reported that nerve growth factor activates CK2α and then stimulates phosphorylation and inhibition of PTEN, and inhibits GSK-3β activity, leading to axon outgrowth (30). Regulation of CK2α activity appears to be a critical step for axon elongation, and activation and inhibition of CK2α lead to axon outgrowth and growth cone collapse, respectively.

We here showed that R-Ras GAP activity suppresses CK2α activity, whereas constitutively active R-Ras promotes PTEN phosphorylation, which is blocked by kinase-dead CK2α, suggesting that R-Ras regulates CK2α activity. At this time, it is unclear how R-Ras regulates CK2α activity. Further work is required to delineate the role of PI3K signaling downstream of plexin-mediated R-Ras GAP activity in a wide range of semaphorin-mediated cellular responses, such as neurite remodeling and cell migration.

Acknowledgments

We thank L. Tamagnone and H. Kikutani for providing Plexin-B1 and the soluble form of Sema4D expression plasmids, respectively.

*This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.

2The abbreviations used are:

Sema
semaphorin(s)
GEF
guanine nucleotide exchange factor
GAP
GTPase-activating protein
LARG
leukemia-associated RhoGEF
myr-
myristoylated
DIV
day(s) in vitro
PIP3
phosphatidylinositol 3,4,5-trisphosphate
bpV(pic)
dipotassium bisperoxo (picolinato) oxovanadate (V).

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