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, 22 (18), 7948-58

A New Activity of Doublecortin in Recognition of the phospho-FIGQY Tyrosine in the Cytoplasmic Domain of Neurofascin

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A New Activity of Doublecortin in Recognition of the phospho-FIGQY Tyrosine in the Cytoplasmic Domain of Neurofascin

Krishnakumar Kizhatil et al. J Neurosci.

Abstract

Doublecortin is a cytoplasmic protein mutated in the neuronal migration disorder X-linked lissencephaly. This study describes a novel activity of doublecortin in recognition of the FIGQY-phosphotyrosine motif present in the cytoplasmic domain of the L1 cell adhesion molecule neurofascin. Phospho-FIGQY-neurofascin (186 kDa) coimmunoprecipitated with doublecortin from detergent extracts of embryonic brain membranes, and this doublecortin-phospho-FIGQY neurofascin complex was disassociated by a synthetic phospho-FIGQY neurofascin peptide but not by a dephospho-FIGQY peptide. Doublecortin specifically recognized the phospho-FIGQY tyrosine in the context of a synthetic phospho-FIGQY neurofascin peptide and in phospho-FIGQY neurofascin isolated from cells treated with pervanadate. Mutations of doublecortin causing lissencephaly (R59H, D62N, and G253D) abolished binding to the phospho-FIGQY peptide and to phospho-FIGQY neurofascin. Finally, phospho-FIGQY neurofascin and doublecortin colocalize in developing axon tracts and in zones enriched in migrating neurons in the embryonic cerebral cortex. In the adult rostral migratory stream, doublecortin colocalizes in migrating neurons with a phospho-FIGQY bearing L1 CAM different from neurofascin. The finding that doublecortin associates with FIGQY-phosphorylated neurofascin provides the first connection of doublecortin with the plasma membrane and could be important for a function of doublecortin in directing neuronal migration.

Figures

Fig. 1.
Fig. 1.
Doublecortin binds directly to a synthetic peptide bearing the phospho-FIGQY motif, and mutations in doublecortin that cause X-linked lissencephaly abolish the doublecortin-phospho-FIGQY motif interaction. A, Coomassie blue-stained gel of purified monodisperse recombinant doublecortin (2 μg).B, Results of a representative peptide-binding assay. The y-axis of the graph represents the doublecortin binding to the synthetic peptides in arbitrary units (A.U.) and was obtained as described in Materials and Methods.C, Coomassie blue-stained gel of partially purified recombinant doublecortin and doublecortin mutants: R59H, D62N, and G253D. D, Results of a representative peptide-binding assay. The y-axis of the graph represents in arbitrary units the doublecortin binding to the synthetic peptides. Black bars represent binding for wild-type doublecortin, white bars represent binding for R59H doublecortin, diagonally striped bars represent binding for D62N doublecortin, andhorizontally striped bars represent binding for G253D doublecortin. In B and D, the peptides used are listed below each bar representing doublecortin binding, phospho–FIGQY, FIGQY, FIGQF, or no peptide.
Fig. 2.
Fig. 2.
Doublecortin and phospho-FIGQY L1 CAMs form a complex in embryonic rat brain.A,Left panel, Immunoblot of equivalent volumes (10 μl) of total embryonic brain homogenate (T), cytosolic fraction (C), and Triton X-100 extract of membrane fraction (M) with a rabbit polyclonal doublecortin antibody. Right panel, Doublecortin in the different fractions (see Materials and Methods) is represented as a percentage of the amount in the starting homogenate (T). B, Immunoblot analysis of immunoprecipitates obtained using doublecortin antibodies from Triton X-100 membrane lysates (Materials and Methods). Antibodies used in immunoprecipitation are shown at thetop of the blots. Antibodies used for immunobloting are shown on the right-hand side of the blots. DCX, Doublecortin; IP, immunoprecipitation. C, Immunoblot analysis of immunoprecipitates obtained using doublecortin antibody as well as total Triton X-100 membrane lysate with anti-NCAM antibody.D, Immunoblot analysis of immunoprecipitates obtained using doublecortin antibody as well as total Triton X-100 membrane lysate with antibodies against neurofascin, L1, and NrCAM. The antibody used for immunoblot is shown on the top of theblots. Tot, Triton X-100 membrane lysate;DCX IP, doublecortin immunoprecipitate.
Fig. 3.
Fig. 3.
Doublecortin-phospho-FIGQY L1 CAM complex in embryonic brain membranes is disrupted by a phospho-FIGQY synthetic peptide but not a dephospho-FIGQY peptide. Shown is immunoblot analysis of immunoprecipitated polypeptides obtained using antibody against doublecortin (Fig. 2) after preincubation of Triton X-100 lysate with synthetic peptides containing either the phospho-FIGQY or the FIGQY motif. The amount and nature of the synthetic peptide used are indicated above the blot. Antibodies used for the immunoblot are shown to the right of the blot.
Fig. 4.
Fig. 4.
Doublecortin binds directly to FIGQY tyrosine-phoshorylated full-length neurofascin. A, Phospho-FIGQY native neurofascin binds doublecortin in the in vitro binding assay (Materials and Methods). B, Mutants of doublecortin that cause neuronal migration defects do not bind to full-length neurofascin. In A andB, the antibodies used for immunoblotting are shown to the right of the blots. The doublecortin proteins used are shown above each of thefour columns of blots. InA and B, pervanadate treatment of the B104 cells transfected with HA-tagged neurofascin before immunoprecipitation is indicated by +, and the lack of treatment is indicated by − above the blots. The type of neurofascin is indicated above the blots inA and B: wt, wild type;Y/F, FIGQY/F neurofascin. In A,none indicates nonspecific mouse IgG used instead of anti-HA antibody. In A, wild-type purified doublecortin was used in all binding sets as indicated above theblots; wt denat refers to heat-denatured wild-type doublecortin. In B the identities of doublecortin proteins used are shown above each of the four columns of blots. C, Graphical representation of the results of the binding assay shown inB. The y-axis represents in arbitrary units the ratio of doublecortin signal to the neurofascin signal after background correction. The neurofascin used in the binding is shown below the x-axis. wt, Wild type;Y/F, FIGQY/F neurofascin. Pervanadate treatment or lack of it is indicated by + or −, respectively. Black barsrepresent binding for wild-type doublecortin, white barsrepresent binding for R59H doublecortin, diagonally striped bars represent binding for D62N doublecortin, andhorizontally striped bars represent binding for G253D doublecortin. DCX, Doublecortin; NF, neurofascin; PV, pervanadate.
Fig. 5.
Fig. 5.
Individual domains of doublecortin fail to bind the phospho-FIGQY motif. A, Coomassie blue-stained gel of purified monodisperse recombinant doublecortin, DCN domain, and DCC domains. B, Peptide binding assay. Thegraph on the left shows the result of assay testing the ability of the DCN domain to bind peptides bearing the phospho-FIGQY motif. Black bars, Doublecortin;gray bars, DCN domain. The graph on theright shows the result of assay testing the ability of the DCC domain to bind peptides bearing the phospho-FIGQY motif.Black bars, Doublecortin; gray bars, DCC domain. Binding is represented as arbitrary units. The peptides used are listed below each bar representing doublecortin binding. C, Binding of native doublecortin and DCN domain to immunoisolated native neurofascin was performed as described (Materials and Methods). The antibodies used for immunoblotting are shown to the right of the blots. The graphical representation of the binding is shown to the right of the blots also. D, Binding of native doublecortin and DCC domain to immunoisolated native neurofascin was performed as described in Materials and Methods. The antibodies used for immunoblotting are shown to the right of theblots. In C and D, the neurofascin used in the binding is also indicated above the blot.wt, Wild type; Y/F, FIGQY/F neurofascin. Pervanadate treatment or lack of it is indicated by + or −, respectively. DCX, Doublecortin; NF, neurofascin; PV, pervanadate. The y-axis represents in arbitrary units the ratio of doublecortin signal to the neurofascin signal after background correction. Black bars, Full-length doublecortin; gray bars, doublecortin domains DCN in C and DCC inD.
Fig. 6.
Fig. 6.
Migrating neurons coexpress phospho-FIGQY L1 CAMs and doublecortin in embryonic brain. A, Double immunofluorescence staining of a parasagittal section of E15 rat brain with anti-phospho-FIGQY antibody and goat anti-doublecortin (Materials and Methods): phospho-FIGQY L1 CAMs (red,left), doublecortin (green,middle), and overlay (right). Scale bar, 10 μm. B, Double immunofluorescence labeling of a parasagittal section of E15 rat brain with rabbit anti-neurofascin antibody and goat anti-doublecortin: neurofascin (red,left), doublecortin (green, middle), and overlay (right). Scale bar, 25 μm. C, Double immunofluorescence labeling of a parasagittal section of E15 rat brain with rabbit anti-doublecortin-13 antibody and goat anti-doublecortin: doublecortin-13 (red, left), doublecortin (green, middle), and overlay (right). Scale bar, 25 μm.
Fig. 7.
Fig. 7.
Migrating neurons in the rostral migratory stream coexpress phospho-FIGQY L1 CAMs and doublecortin in the adult brain. A, Doublecortin and phospho-FIGQY L1 CAMs are coexpressed in migrating neurons of the rostral migratory stream in adult brain. Shown is double immunofluorescence labeling of a parasagittal section of the rostral cortex of adult rat brain using rabbit anti-phospho-FIGQY antibody and goat anti-doublecortin: phospho-FIGQY L1 CAM (red, left), doublecortin (green,middle), and overlay (right).Arrows indicate the chain-like structures formed by the migrating neurons. Scale bar, 50 μm. B, Doublecortin and phospho-FIGQY L1 CAMs are coexpressed in primary cultures of SVZ neurons. Cultures (Materials and Methods) were subjected to immunofluorescence with anti-phospho-FIGQY L1 CAM antibody, goat anti-doublecortin, and monoclonal anti-βIII tubulin. Top panels show the DIC image (left) and βIII tubulin (right); bottom panels show phospho-FIGQY L1 CAM (red, left), doublecortin (green, middle), and overlay (right). βIII tubulin-positive chain migrating neurons (arrowheads): non-neuronal process as indicated by the lack of βIII tubulin (arrow). Scale bar, 25 μm.
Fig. 8.
Fig. 8.
Doublecortin and phospho-FIGQY L1 CAMs colocalize in axon fascicles during development. Double immunofluorescence of sections of rat tissue was performed with anti-phospho-FIGQY antibody and goat anti-doublecortin antibody. Phospho-FIGQY L1 CAM (a, d,g, red), doublecortin (b,e, h, green), and overlap (c, f, i).ac, Axon tracts in the dorsal root ganglion; overlap (c). DCX, Doublecortin; DRG, dorsal root ganglion;phospho-FIGQY, phospho-FIGQY L1 CAM. Scale bar, 100 μm. df, Axon fascicle (arrows) in olfactory placode.axf, Axon fascicle; nb, neuroblastoma; ob, developing olfactory bulb;op, olfactory placode; vno, vomeronasal organ. Scale bar, 100 μm. gi, Axon fascicle (arrow) in the developing mandible. Scale bar, 50 μm.

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