CRMP2 is phosphorylated by Fyn in the region of 1–212. A, identification of the region of CRMP2 phosphorylated by Fyn. CRMP2 full-length or CRMP2 partial fragment 1–212, 231–572, or 415–572 was transfected into HEK293T cells with FynCA. The fragments were immunoprecipitated (IP) and subjected to immunoblotting with anti-phosphotyrosine antibody (PY-99) or anti-Myc antibody. The N-terminal region of CRMP2-(1–212) was well phosphorylated by Fyn. Asterisks indicate phosphorylated or intact bands of CRMP2. Arrowheads indicate nonspecific bands. The two bands in Myc blot of CRMP2-(213–572) and CRMP2-(415–572) may be caused by proteolysis. B, summary of the results shown in A.

In vitro phosphorylation of CRMP2wt-(1–69) and CRMP2Y32F-(1–69). A, alignment of amino acid sequences of rat CRMP1–5. Sequence of rat CRMP1–5, amino acids 25–50. Black line indicates that the epitope of the functional blocking antibody (1) Tyr³² is included in this region. B, in vitro phosphorylation of CRMP2wt-(1–69) or the CRMP2Y32F-(1–69) mutant. In vitro kinase assay was performed using purified Fyn and CRMP2wt-(1–69) or CRMP2Y32F-(1–69) with or without ATP (0.5 mm). Each reaction mixture was subjected to SDS-PAGE and immunoblot analysis with anti-phosphotyrosine antibody (4G10). The same samples were separated and stained by Coomassie Brilliant Blue (CBB) to show that relatively similar amounts of CRMP2 were loaded.

Imunohistochemical analysis with anti-pCRMP (Y32) antibody. A, immunohistochemistry with anti-pCRMP (Y32) antibody (green), anti-NeuN antibody or anti-Tuj1 antibody, a marker for neurons (red), in the coronal sections around spinal cord and DRG at E13.5. TO-PRO-3 iodide was used for nuclei staining (blue). Preimmunized serum was used for the negative control. Arrows show DRG and spinal cord. Scale bars, 100 μm. SC, spinal cord. B, immunohistochemistry with anti-pCRMP (Y32) antibody (green) with anti-NeuN antibody (red) in the sagittal sections of hippocampus at E18.5. TO-PRO-3 iodide was used for nuclei staining (blue). The arrow shows hippocampal fimbria. Scale bar, 100 μm. C, immunohistochemistry with anti-pCRMP (Y32) antibody (green) with anti-calbindin (CB) antibody, a marker for Purkinje cells (red), in the sagittal sections of the cerebellum at 4-week-old wt mouse. TO-PRO-3 iodide was used for nuclei staining (blue). Low magnification and high magnification images are shown. The highest level of the phosphorylation was seen in the Purkinje cell layer and molecular layer (arrow). ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, 200 μm (low magnification) and 50 μm (high magnification).

CRMP family proteins are phosphorylated by Fyn in HEK293T cells. Each CRMP family member was transfected into HEK293T cells with or without FynCA. Myc-epitope tagged CRMPs were immunoprecipitated (IP) with anti-Myc antibody and immunoblotted with anti-phosphotyrosine antibody (top). The same membrane was reprobed by anti-Myc antibody (middle). The expression of FynCA in the cell lysates was confirmed by the immunoblotting with anti-Fyn antibody (bottom). All members of the CRMP family (CRMP1–5) are tyrosine-phosphorylated in the presence of Fyn (top).

Tyr³² is a common phosphorylation site of CRMP2 by Fyn and Fes tyrosine kinases. A, prediction of the phosphorylation sites of rat CRMP2 using the Web-based program NetPhos (20). This program proposes the tyrosine phosphorylation sites of CRMP2 at Tyr³², Tyr³⁶, Tyr¹⁸², Tyr²⁵¹, Tyr²⁹⁰, and Tyr⁴⁷⁹. B, Tyr³² is the phosphorylation site of Fyn. The nonphosphorylated mutants, CRMP2Y32F, Y36F, Y182F, Y479F, Y32F/Y36F, and Y32F/Y182F were generated by PCR-based mutagenesis. These mutants or CRMP2wt were cotransfected with FynCA into HEK293T cells. The cell lysates were immunoprecipitated (IP) with anti-Myc antibody. The samples were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (PY-99). The same membrane was reprobed with anti-Myc antibody. To confirm expression of FynCA, the cell lysates were immunoblotted with anti-Fyn antibody. The phosphorylation level of the CRMP2Y32F mutant was decreased compared with that of wt. The quantitative data include mean values ± S.E. for n = 3. **, p < 0.01, significantly different from CRMP2wt using analysis of variance. C, Fes phosphorylates CRMP2 at Tyr³² and Tyr¹⁸². The mutants or CRMP2wt was cotransfected with Fes-GFP into HEK293T cells. The cell lysates were immunoprecipitated with anti-Myc antibody. The samples were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (PY-99). The same membrane was reprobed with anti-Myc antibody. To confirm expression of Fes-GFP, the cell lysates were immunoblotted with anti-GFP antibody. The quantitative data include mean values ± S.E. for n = 3. *, p < 0.05 and **, p < 0.01, significantly different from CRMP2wt using analysis of variance.

Tissue distribution of phosphorylated CRMP at Tyr³². A, specificity of phospho-specific antibody for CRMP2. The specificity of anti-pCRMP (Y32) antibody was examined with immunoblot analysis. Purified CRMP2wt-(1–69) or CRMP2Y32F-(1–69) was subjected to a phosphorylation assay using Fyn, separated by SDS-PAGE, and immunoblotted with anti-pCRMP (Y32) antibody. This antibody recognizes phosphorylated CRMP2 but not nonphosphorylated CRMP2. The phosphorylation levels of CRMP2 increase in a time-dependent manner. The same samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) to show the amount of CRMP2 loaded. B, phosphorylation of CRMP1, CRMP2, and CRMP4 at Tyr³² by Fyn in HEK293T cells. HEK293T cells were transfected with CRMP1–5 alone or together with FynCA. The cell lysates were immunoprecipitated (IP) with anti-Myc antibody and then immunoblotted with anti-pCRMP (Y32) antibody and Myc antibody. This anti-pCRMP (Y32) antibody recognizes the phosphorylated form of CRMP2, CRMP4, and weakly CRMP1. C, developmental changes in the levels of the phosphorylated CRMP2 at Tyr³² in spinal cord. Spinal cord lysates (10 μg) at several developmental stages were immunoblotted with anti-pCRMP (Y32) antibody and anti-CRMP2 antibody (C4G). Black arrowheads indicate CRMP2. The multiple bands may be due to cross-reaction of the anti-pCRMP (Y32) antibody against the phosphorylated form of CRMP2 and CRMP4. D, developmental changes in the levels of the phosphorylated CRMP2 at Tyr³² in brain. Whole brain lysates (20 μg) at several developmental stages were immunoblotted with anti-pCRMP (Y32) antibody and anti-CRMP2 antibody (C4G). Black arrowheads indicate CRMP2. Open arrowheads indicate Ser/Thr-phosphorylated form of CRMP2. E, lysates (20 μg) of the cortex, hippocampus, cerebellum, brain stem, and olfactory bulb of adult mouse were immunoblotted with anti-pCRMP (Y32) antibody. The level of phosphorylated CRMP at Tyr³² was highest in the cerebellum. Black arrowheads indicate CRMP2. Open arrowhead indicates Ser/Thr-phosphorylated form of CRMP2.

Sema3A-induced CRMP2 phosphorylation at Tyr³² and suppression of Sema3A-induced growth cone collapse by introduction of the nonphosphorylated mutant. A, COS-7 cells were transfected with PlexinA2, NRP1, Fyn, and CRMP2-Myc. After being stimulated with 3 nm Sema3A, the cell lysates were immunoblotted with anti-pCRMP (Y32) antibody and anti-Myc antibody. A typical immunoblot analysis is shown. B, chick E7 DRG neurons were infected with recombinant herpes simplex virus preparations directing the expression of CRMP2wt or the CRMP2Y32F mutant. Expression of CRMP2wt did not alter Sema3A-induced growth cone collapse, whereas expression of CRMP2Y32F suppressed the Sema3A response. Scale bar, 20 μm. C, rate of growth cone collapse of CRMP2wt- or CRMP2Y32F-expressing DRG neurons stimulated with Sema3A (0.5 nm). Data include mean values ± S.E. for n = 9–13. *, p < 0.01, significantly different from no virus control using analysis of variance.