Nck beta interacts with tyrosine-phosphorylated disabled 1 and redistributes in Reelin-stimulated neurons - PubMed (original) (raw)

Nck beta interacts with tyrosine-phosphorylated disabled 1 and redistributes in Reelin-stimulated neurons

Albéna Pramatarova et al. Mol Cell Biol. 2003 Oct.

Abstract

The tyrosine phosphorylation sites of the Disabled 1 (Dab1) docking protein are essential for the transmission of the Reelin signal, which regulates neuronal placement. Here we identify Nck beta as a phosphorylation-dependent, Dab1-interacting protein. The SH2 domain of Nck beta but not Nck alpha binds Dab1 phosphorylated on the Reelin-regulated site, Y220, or on Y232. Nck beta is coexpressed with Dab1 in the developing brain and in cultured neurons, where Reelin stimulation leads to the redistribution of Nck beta from the cell soma into neuronal processes. We found that tyrosine-phosphorylated Dab1 in synergy with Nck beta disrupts the actin cytoskeleton in transfected cells. In Drosophila melanogaster, exogenous expression of mouse Dab1 causes tyrosine phosphorylation site-dependent morphological changes in the compound eye. This phenotype is enhanced by overexpression of the Drosophila Nck protein Dock, suggesting a conserved interaction between the Disabled and Nck family members. We suggest a model in which Dab1 phosphorylation leads to the recruitment of Nck beta to the membrane, where it acts to remodel the actin cytoskeleton.

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Figures

FIG. 1.

FIG. 1.

Dab1 tyrosine phosphorylation and the Nckβ SH2 domain are required for complex formation. Nck proteins were detected by anti-HA Western blotting (upper panel) of cell lysates from HEK293T cells transfected with Nckα (lanes 1, 2, and 14), Nckβ (lanes 3, 4, 7 to 13, and 15), Nckβ-R312K carrying a mutation in the SH2 domain (lanes 5 and 16), or Nckβ-W39,149,235K, which has mutations in all three SH3 domains (lanes 6 and 17). The proteins that bound the various GST-Dab1 fusion proteins were eluted by phenyl phosphate-containing buffer (lanes 4 to 17; upper panel). The GST-Dab1 fusions included Dab1-wt (lanes 1 to 6, 12, and 13), Dab1-Y185F (lane 7), Dab1-Y198,200F (lane 8), Dab1 Y220F (lane 9), Dab1 Y232F (lane 10), and Dab1 Y220,232F (lane 11) in their unphosphorylated state (Y; lanes 1, 3, and 13) or after tyrosine phosphorylation by Abl (pY or mutant designation, lanes 2 and 4 to 12). All GST fusion proteins incubated with Abl kinase were tyrosine phosphorylated as determined by Western blotting with antiphosphotyrosine antibody 4G10 (lower panel). Lanes 14 to 17 represent 1% of the amount of cell lysate used in the association assay (lanes 1 to 13).

FIG. 2.

FIG. 2.

Nckβ coimmunoprecipitates with tyrosine-phosphorylated Dab1 but not the unphosphorylated Dab1-5F mutant. Immunoprecipitations with anti-Dab1 or anti-HA antibody, as indicated to the left of the panels, were done from lysates of HEK293T cells transfected with SrcY527F (lanes 1 to 5) and combinations of Nckβ (lanes 1, 3, and 5) and Dab1 wild-type (lanes 2 and 3) or Dab1-5F (lanes 4 and 5). Immunoprecipitation of Dab1 with an anti-C-terminal antibody followed by Western blotting with anti-Dab1 (B3) (top panel) shows that transfected cells express at least as much mutant Dab1-5F as wild-type Dab1, but immunoblotting with antiphosphotyrosine antibody (4G10; second panel) shows that only the wild-type Dab1 was tyrosine phosphorylated. Equal amounts of Nckβ were immunoprecipitated from all HA-Nckβ-transfected cells (third panel, lanes 1, 3, and 5), yet Dab1 was only detected in anti-HA immunocomplexes (bottom panel) from cells expressing both Nckβ and wild-type Dab1 (lane 3).

FIG. 3.

FIG. 3.

Nckβ is expressed in Dab1-expressing Reelin-responsive cells. (A) The Nckβ protein is detected in the cell bodies of Purkinje cells, which also express Dab1 (B) in the brains of adult mice. (C) Both Dab1 and Nckβ are absent from the internal granule cell layer, which is apparent below the Purkinje cells with the nuclear indicator DAPI. Bar, 10 μm.

FIG. 4.

FIG. 4.

Nckβ is redistributed into neuronal processes after Reelin stimulation. (A and B) In cultures treated with control conditioned medium for 30 min, Nckβ (A; green) is found predominantly in the cell soma. (C and D) After Reelin treatment, Nckβ (C; green) is apparent in the majority of processes. Processes were detected (B and D) with fluorescently labeled phalloidin to visualize filamentous actin (red), and nuclei were detected with DAPI. Bar, 20 μm.

FIG. 5.

FIG. 5.

Nckβ colocalizes with Dab1 in process termini after Reelin stimulation. (A to C) Nckβ (green; A and C) is predominantly found in the soma of primary neurons from Reelin mutant animals fixed after 10-min treatments with control conditioned medium, while Dab1 (red; B and C) was found throughout the neuron. Colocalization between Nckβ and Dab1 was observed in the cell soma but not in the processes (yellow; C). (D to F) After treatment with Reelin-conditioned medium for 10 min prior to fixation, Nckβ (D and F) was detected in processes with Dab1 (E and F), and colocalization (F) was apparent near the process termini. Bar, 20 μm.

FIG. 6.

FIG. 6.

Mouse Dab1RFP fusion is tyrosine-phosphorylated when expressed in Rat-2 cells and fly eyes. (A) Cell lysates from Rat-2 fibroblasts transfected with Dab1GFP (lanes 1 and 4), Dab1-wt (lanes 2 and 5), or Dab1RFP (lanes 3 and 6) were immunoprecipitated with anti-Dab1 C-terminal antibody. Western blotting with anti-Dab1 antibody (lanes 1 to 3) showed that comparable levels of Dab1 were expressed, but in the antiphosphotyrosine Western blot (lanes 3 to 6), only Dab1RFP (lane 6) was detected. (B) Expression of Dab1RFP and Dab1RFP-5F was detected in total cell lysates of flies with the genotypes UAS-Dab1RFP/GMR-GAL4 (lane 1), UAS-Dab1RFP-5F/GMR-GAL4 (lane 2), and UAS-Dab1RFP-5F;UAS-Dab1RFP-5F/GMR-GAL4 (lane 3) to compare the levels of expression of the wild-type and Dab1-5F mutant flies as adults. Expression of both of these protein products was also observed by Western blot in wandering third-instar larvae and pupae (not shown). Lysates from UAS-Dab1RFP/GMR-GAL4 (lane 4) and UAS-Dab1RFP-5F/GMR-GAL4 (lane 5) flies were immunoprecipitated with anti-Dab1 antibody (Chemicon) and Western blotted for antiphosphotyrosine (4G10; Upstate Biotechnology), showing that the Dab1RFP fusion but not the Dab1RFP-5F protein is tyrosine phosphorylated when expressed in the adult fly. The same was observed for wandering third-instar larvae and pupae (not shown).

FIG. 7.

FIG. 7.

Nckβ colocalizes with Dab1 at the cell periphery of Rat-2 fibroblasts in a tyrosine phosphorylation site-dependent manner. (A to C) Dab1RFP (A and C; red) is detected in an intense peripheral ring in the majority of expressing Rat-2 cells, and Nckβ (B and C; green) is observed to colocalize in this region. (D to F) Dab1RFP-5F (D and F) is expressed in a pattern similar to the wild-type counterpart, however, Nckβ (E and F) does not colocalize at the cell periphery. (G to I) Native Dab1 (G and I) is predominantly cytoplasmic, and Nckβ (H and I) is also diffuse throughout the cytoplasm, with no concentration near the plasma membrane. Bar, 20 μm.

FIG.8.

FIG.8.

Coexpression of tyrosine-phosphorylated Dab1RFP and Nckβ in Rat-2 cells leads to the disruption of the actin cytoskeleton. The actin cytoskeleton, detected with fluorescently labeled phalloidin (white; B, D, F, H, J, and L) observed on the lower plane of Rat-2 fibroblasts transfected with HA-Nckβ (green; A and B), or Dab1RFP, a tyrosine-phosphorylated form of Dab1 (red; C and D), shows the typical mesh pattern. Combined expression of HA-Nckβ and Dab1RFP (E and F) leads to disruption of the actin mesh and clumping of actin filaments. Expression of unphosphorylated Dab1-wt with HA-Nckβ (G and H), or Dab1RFP-5F with HA-Nckβ (I and J) did not disrupt the actin cytoskeleton, nor did expression of Dab1RFP with the SH2 domain mutant of Nckβ (K and L). All images show the plane of the cell closest to the cover glass. Bar, 20 μm.

FIG. 9.

FIG. 9.

Nck-enriched regions in distal processes of Reelin-treated neurons have patterns of actin filaments not observed in control-treated neurons. (A and B) The growth cones of neurons at 1 day in vitro demonstrate intense actin filaments (arrow) detected by phalloidin staining (A and B; red and white) and were devoid of Nckβ (green; A). (C to F) Approximately 5% of neurons had Nckβ enrichments in distal processes 10 min after Reelin stimulation (arrowheads). The Nckβ-enriched regions had patterns of actin filaments that varied from actin-poor (C and D) to intense peripheral actin features such as actin rings (star; E and F). Bar, 20 μm.

FIG. 10.

FIG. 10.

Overexpression of Dock in the D. melanogaster eye enhances a phosphorylation site-dependent Dab1RFP phenotype. Flies with the genotypes UAS-RFP/GMR-GAL4 (A), UAS-Dab1RFP/GMR-GAL4 (B), UAS-Dab1RFP-5F/GMR-GAL4 (C), UAS-Dock; UAS-RFP/GMR-GAL4 (D), UAS-Dock; UAS-Dab1RFP/GMR-GAL4 (E), and UAS-Dock; UAS-DabRFP-5F/GMR-GAL4 (F) were examined by scanning electron microscopy. Representative phenotypes are shown from over 10 examples of each genotype that were examined. Expression of the RFP proteins was confirmed by fluorescence microscopy (data not shown). Magnification: ×200 (scale bar, 100 μm). Inset magnification: ×1,000.

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