Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones

Abstract

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.

FIG. 1.

An in vitro tubulin or microtubule binding assay using phosphorylated or nonphosphorylated CRMP-2-GST. (A) GST and CRMP-2-GST (0.5 μM) were phosphorylated by Rho kinase catalytic domain (RhoK-cat) in the presence (+) or absence (−) of ATP. GST or CRMP-2-GST immobilized onto beads was incubated with 1 μM tubulin in PEM buffer for 1 h at 4°C. GST and CRMP-2-GST results were shown with a Coomassie brilliant blue (CBB)-stained gel (right panel). Purified CRMP-2-GST contained degradation products, as confirmed by immunoblotting (IB) with anti-GST and anti-CRMP-2 antibody. An asterisk indicates RhoK-cat. An arrowhead and an arrow indicate the intact protein of CRMP-2-GST and GST, respectively. Phosphorylated CRMP-2-GST or the bound tubulin was analyzed by immunoblotting with anti-phospho-CRMP-2 antibody and anti-α-tubulin antibody (left panels). (B) Biacore sensorgram of tubulin capture with anti-tubulin antibody. Purified tubulin heterodimer (0.5 μM) was captured with the anti-α-tubulin antibody immobilized over the sensor chip at a flow rate of 10 μl/min for 3 min. The sensorgram in the sample of nonphosphorylated CRMP-2 [ATP (−)] is identical to that obtained with phosphorylated CRMP-2 [ATP (+)] (data not shown). The increase in RU (resonance units) results from the binding of tubulins to anti-tubulin antibodies. (C) Biacore sensorgram of CRMP-2 binding to tubulin. Binding of 8, 4, 2, 1, and 0.5 μM phosphorylated [ATP (+)] or nonphosphorylated [ATP (−)] His-CRMP-2 to a tubulin heterodimer-loaded sensor chip surface was examined. (D) Cosedimentation analysis of phospho-CRMP-2 or non-phospho-CRMP-2. His-CRMP-2 (5 μM) was phosphorylated by Rho kinase (RhoK-cat) in the presence (+) or absence (−) of ATP. His-CRMP-2 was mixed with 10 μM microtubules stabilized by Taxol (+) or left unmixed (−). After a 10-min incubation at 37°C, mixtures containing microtubules were centrifuged at 37°C. The quantity of His-CRMP-2 in supernatant (S) or pellet (P) was shown by Coomassie brilliant blue gel staining results (upper panel). The samples were analyzed by immunoblotting using anti-GST antibody to confirm the identity of this band as CRMP-2 (lower panel). An asterisk indicates RhoK-cat. An arrowhead and an arrow indicate His-CRMP-2 and tubulin, respectively.

FIG. 2.

An in vitro binding assay using phospho- or non-phospho-CRMP-2-GST and rat brain lysate. (A and B) GST and CRMP-2-GST (0.5 μM) were phosphorylated by Rho kinase catalytic domain (RhoK-cat) (A) or Cdk5 and/or GSK-3β (B) in the presence (+) or absence (−) of ATP. GST and CRMP-2-GST immobilized on beads were incubated with extracts of rat brain (P6 and P7) for 1 h at 4°C. CRMP-2-GST and the bound proteins were analyzed by immunoblotting (IB) using anti-GST antibody, anti-phospho-CRMP-2 antibody, anti-α-tubulin antibody, anti-Numb antibody, anti-Rho-GDI antibody, or anti-actin antibody. Input, immunoreactive bands of brain lysate (5% in total lysate). The lane labeled “beads only” shows the results for beads and brain lysates without GST proteins.

FIG. 3.

Localization of CRMP-2 or phospho-CRMP-2 in chick DRG neuron growth cones. (A) Chick DRG neuron growth cones were triple stained with anti-CRMP-2 antibody (red), anti-unique β-tubulin antibody (green), and Alexa-649-phalloidin (blue) (upper panels) or anti-phospho-CRMP-2 antibody (red), anti-unique β-tubulin antibody (green), and Alexa-488-phalloidin (blue) (lower panels). Arrowheads indicate the colocalization of phosphorylated CRMP-2 or CRMP-2 and actin filaments. (B) Graphs plot the fluorescence intensity of immunolabeled CRMP-2 (red) and unique β-tubulin (green) or phosphorylated CRMP-2 (red) and unique β-tubulin (green) in the dotted line shown in each growth cone image. Bar, 10 μm.

FIG.4.

Electron microscopic immunocytochemical localization of CRMP-2 in chick DRG growth cones. (A to C) Horizontal section of central area of growth cones. Enlarged images from insets a to c in panels A to C are shown in panels a to c. In panels a and b, red arrows indicate the immunolabeling of CRMP-2. Thin lines are microtubule filaments. (B) Grazing section of growth cones. Red arrows indicate the immunolabeling of CRMP-2 close to the plasma membrane. (C) Immunolabeling with anti-phospho-CRMP-2 was not observed. Bars (panels a to c), 500 nm. (D to H) High-power view of the sample prepared by freeze-etching immunoreplica methods (negative images are shown for clarity). Immunocolloidal gold is shown as white dots. Red arrows indicate the immunolabeling of CRMP-2 on clathrin-coated pits (D and E) and on actin filaments (G) or of phosphorylated CRMP-2 on actin filaments (H). Immunolabeling with anti-phospho-CRMP-2 antibody was not observed on clathrin-coated pits (F). Bar (D to H), 50 nm.

FIG. 5.

Effects of CRMP-2 mutants on neurite formation in N1E-115 cells and DRG neurons. (A) Localization of GFP-tagged CRMP-2 and CRMP-2 mutants T555A and T555D. Vero cells were transfected with pEGFP-CRMP-2 WT, T555A, or T555D and cultured for 36 h. Transfected cells were fixed and stained with anti-α-tubulin antibody. Arrows indicate the colocalization of GFP-fused protein with microtubules. Bar, 10 μm. (B) The effect of overexpression of CRMP-2 WT, T555A, and T555D on process formation in N1E-115 cells. N1E-115 cells were transfected with myc-GST (as a negative control), CRMP-2 WT, T555A, or T555D. Twenty-four hours after transfection, the cells were cultured in serum-containing medium for 24 h. Transfected cells were fixed and doubly stained by anti-myc antibody (top panels) and anti-α-tubulin antibody (bottom panels). Arrows indicate the processes induced by CRMP-2 constructs. Bar, 40 μm. (C) The percentage of cells bearing neurites (length > 20 μm) in transfected cells. Fifty randomly selected transfected neurons were quantified for each construct. The values shown are means ± standard errors of triplicate experiments. **, significantly different from the cells expressing GST as analyzed by Student's t test (P < 0.01). (D) The percentage of cells bearing axons (length > 1,300 μm) in DRG neuron-transfected cells. DRG neurons were transfected with the plasmids indicated in panel B. After transfection, the neurons were cultured in the NGF-deprived medium for 3 days. Transfected neurons were fixed and doubly stained by anti-myc antibody and anti-neurofilament antibody. The percentage of neurons bearing axons with length > 1,300 μm versus the total number of neurons bearing axons was analyzed. Fifty randomly selected transfected neurons were quantified for each construct. The values shown are means ± standard errors of triplicate experiments. **, significantly different from the cells expressing GST as analyzed by Student's t test (P < 0.01); *, P < 0.05.

FIG. 6.

Ephrin-A5-induced CRMP-2 phosphorylation. (A) DRG neurons were serum starved for 4 h and stimulated by 1 μg/ml ephrin-A5 with or without pretreatment with 10 μM Rho kinase inhibitor (Y-27632 or HA1077) for 0, 3, 10, and 30 min. The cell lysate was resolved by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. The multiple bands of CRMP-2 represent differentially phosphorylated forms (33). Arrowheads indicate the intact protein of CRMP-2. The band corresponding to the phosphorylated form by Rho kinase was detected at the same position as the main band detected by anti-CRMP-2 antibody. The results are representative of three independent experiments. (B) The relative levels resulting from CRMP-2 phosphorylation were calculated, with the level obtained with untreated control cells defined as 100 units. (C) The localization of phosphorylated CRMP-2 before and after stimulation with ephrin-A5 in DRG neurons. DRG neurons were serum starved for 4 h and stimulated by 1 μg/ml ephrin-A5. Before stimulation and 10 min after stimulation with ephrin-A5, cells were fixed and immunostained with anti-phospho-CRMP-2 antibody (red) and anti-CRMP-2 antibody (green). Arrows indicate the collapsed growth cone and axonal shaft, which was intensely stained by anti-phospho-CRMP-2 antibody. Bar, 20 μm. The fluorescence intensities of CRMP-2 (green) and phospho-CRMP-2 (red) in the dotted lines are shown in the graphs (bottom panel). Arrows indicate the same positions as the areas indicated by arrows in the images (upper panels). (D) Effects of CRMP-2 mutants T555A and T555D on ephrin-A5-induced growth cone collapse. DRG neurons transfected with myc-GST, CRMP-2 WT, T555A, or T555D were stimulated with ephrin-A5 for 30 min. Then the population of collapsed growth cones expressing introduced constructs was calculated. Thirty randomly selected transfected neurons were quantified for each construct. The values shown are means ± standard errors of triplicate experiments. *, significantly different from the growth cones expressing GST as analyzed by Student's t test (P < 0.05).

FIG. 7.

Model schema for the phosphorylation of CRMP-2 by Rho kinase, Cdk5, and GSK-3β. Sema3A is thought to activate Cdk5 and GSK-3β. These activations cause phosphorylation at Ser-522, Ser-518, and Thr-514. Ephrin-A5 stimulation activates the Rho/Rho kinase signaling pathway and subsequently induces the phosphorylation of CRMP-2 at Thr-555 by Rho kinase. The binding activity of CRMP-2 to tubulin is decreased by the phosphorylation by Cdk5, GSK-3β, and Rho kinase. Nonphosphorylated CRMP-2 binds to tubulin heterodimers to promote microtubule assembly or Numb-mediated endocytosis, thereby enhancing axon elongation and branching. In contrast, the phosphorylated form cannot associate with interacting molecules and loses the positive effect on axon elongation, thereby causing arrest of axon growth and growth cone collapse.