EphA4 Induces the Phosphorylation of an Intracellular Adaptor Protein Dab1 via Src Family Kinases

Abstract

Dab1 is an intracellular adaptor protein essential for brain formation during development. Tyrosine phosphorylation in Dab1 plays important roles in neuronal migration, dendrite development, and synapse formation by affecting several downstream pathways. Reelin is the best-known extracellular protein that induces Dab1 phosphorylation. However, whether other upstream molecule(s) contribute to Dab1 phosphorylation remains largely unknown. Here, we found that EphA4, a member of the Eph family of receptor-type tyrosine kinases, induced Dab1 phosphorylation when co-expressed in cultured cells. Tyrosine residues phosphorylated by EphA4 were the same as those phosphorylated by Reelin in neurons. The autophosphorylation of EphA4 was necessary for Dab1 phosphorylation. We also found that EphA4-induced Dab1 phosphorylation was mediated by the activation of the Src family tyrosine kinases. Interestingly, Dab1 phosphorylation was not observed when EphA4 was activated by ephrin-A5 in cultured cortical neurons, suggesting that Dab1 is localized in a different compartment in them. EphA4-induced Dab1 phosphorylation may occur under limited and/or pathological conditions in the brain.

INTRODUCTION

Dab1 is an intracellular adaptor protein essential for brain formation and development.¹⁾ Dab1 deficiency severely impairs neuronal migration and lamination in the neocortex, hippocampus, and cerebellum.^2–6) Recently, the loss of Dab1 was found to be closely associated with the onset of neuropsychiatric disorders. Forebrain-specific Dab1-conditional knockout mice exhibit behavioral abnormalities similar to those observed in patients with schizophrenia and bipolar disorder.⁷⁾ Furthermore, the biallelic loss of Dab1 has been found in patients with lissencephaly.⁸⁾ Therefore, elucidating the functional regulation of Dab1 is necessary to understand brain development and the pathogenesis of neuropsychiatric disorders.

Dab1 is phosphorylated by Src family kinases (SFKs), such as Fyn,^9,10) which regulates the function of various downstream molecules in a phosphorylation-dependent/independent manner. Dab1 is required for neuronal positioning, dendritic development, and synaptic plasticity during brain development.^2,11) The most well-known regulatory mechanism of Dab1 phosphorylation is a signaling pathway initiated by Reelin and its receptors. Reelin is a secreted glycoprotein that binds to apolipoprotein E receptor 2 (ApoER2) and very low-density receptor (VLDLR). This interaction induces tyrosine phosphorylation in Dab1.¹¹⁾ The five tyrosine residues of Dab1 (Tyr185, 198, 200, 220, and 232) are potentially phosphorylated due to the activation of SFKs.¹²⁾ Notably, phosphorylated Dab1 has been detected in the neocortex of Reelin-deficient mice,²⁾ suggesting that upstream cues other than Reelin induce Dab1 phosphorylation. Recently, netrin1-deleted in colorectal cancer,¹³⁾ epidermal growth factor-epidermal growth factor receptor,¹⁴⁾ and vascular endothelial growth factor-vascular endothelial growth factor receptor¹⁵⁾ were reported to mediate Dab1 phosphorylation in cortical neurons, cancer cells, and endothelial cells, respectively. These findings suggested that Dab1 phosphorylation is regulated by various non-canonical mechanisms in different cells.

In this study, we searched for a molecule that can induce Dab1 phosphorylation and identified EphA4, a member of Eph receptor tyrosine kinases, as bearing such activity. EphA4 is a well-studied Eph family protein and is autophosphorylated by interacting with its ligands, ephrin-A, and plays important roles in cell migration, axonal guidance, and synaptic plasticity.¹⁶⁾ We found that the activation of the cytoplasmic domain of EphA4 and SFKs are required for Dab1 phosphorylation. However, ephrin-A5 stimulation of cultured cortical neurons did not lead to Dab1 phosphorylation. Our results suggest that EphA4 is a novel molecule that can induce Dab1 phosphorylation, and that its contribution to the regulation of Dab1 function may occur under specific conditions.

MATERIALS AND METHODS

Animals

All experimental protocols used in this study were approved by the Animal Care and Use Committee of Nagoya City University and performed according to the Institutional Guidelines on Animal Experimentation of Nagoya City University (Approval Number: 23-001H03). Mice were housed under a 12-h light/dark cycle with lights on between 6:00 a.m. and 6:00 p.m., with standard diets and water ad libitum. Crl:CD-1 mice (MGI Cat# 5652673; RRID: MGI:5652673) were obtained from Japan SLC. Reelin-deficient mice (B6C3Fe a/a-Reln^rl/J, RRID:IMSR_JAX:000235) were obtained from Jackson Laboratory and backcrossed with the Crl:CD-1 strain. Both male and female mice were used in all experiments.

Expression Plasmids

The expression plasmids for VLDLR-green fluorescent protein (GFP),¹⁷⁾ ApoER2-GFP,¹⁷⁾ amyloid precursor protein (APP),¹⁸⁾ ephrin-B1,¹⁸⁾ FLAG-EphA3,¹⁸⁾ and EphB2¹⁸⁾ have been previously described. The vector pcDNA3.1-FLAG-EphA4 (kindly provided by Prof. J. G. Flanagan, Harvard Medical School) was used to express full-length EphA4 (EphA4-WT) and as a template for PCR. The PCR products encoding the K653M or Y596/602F mutations were obtained by overlap-extension PCR and cloned into the EcoRV/XhoI sites of pcDNA3.1-FLAG-EphA4. The expression plasmids for ephrin-A5-Fc and ephrin-B1-Fc were kindly provided by Prof. J. G. Flanagan. The expression plasmids for Dab1-HA and Dab1-5F-HA were kindly provided by Prof. K. Nakajima (Keio University School of Medicine). The expression plasmids for Reelin¹⁹⁾ and Neuropilin-1 (Nrp1)-GFP were kindly provided by Prof. J. Takagi (Osaka University). All the constructs were confirmed by DNA sequencing. Detailed maps of all the expression plasmids are available upon request.

Cell Culture and Transfection

HEK293T and COS-7 cells were cultured as previously described.^20,21) Primary cultured cortical neurons were prepared from embryonic day 16.5 mice and cultured as previously described.^20,21) Transfection was performed using Polyethylenimine “Max” (Polysciences) according to the manufacturer’s instructions. To collect the supernatant containing human Fc-fused proteins, we used the Expi293 expression system (Thermo Fisher Scientific, Waltham, MA, U.S.A.) as previously described.²²⁾ Culture supernatants were collected 72 h after transfection, and debris was removed using a 0.45-µm filter membrane. Fc-fused proteins were purified using Protein G Sepharose and their concentrations were quantified using Protein Assay Bradford Regents (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), as previously described.¹⁸⁾ For the inhibition of SFKs, COS-7 cells were incubated with culture medium containing 5 µM PP2 (Cayman chemical, Ann Arbor, MI, U.S.A., #13198) or 5 µM PP3 (Cayman chemical, #15595) for 5 h.

Immunoprecipitation, Sodium Dodecyl Sulfate-Gel Electrophoresis (SDS-PAGE), and Western Blotting

Immunoprecipitation, sodium SDS-PAGE, and Western blotting were performed as previously described.^18,20) For Western blotting, the antibodies were diluted in 2% skim milk in Tris-buffered saline containing 0.05% Tween 20 or Can Get Signal reagent (Toyobo, Osaka, Japan). Chemiluminescence signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma) and captured using an LAS 4000mini (FUJIFILM, Tokyo, Japan).

Immunostaining

Immunostaining was performed as previously described.^18,20) To induce Eph phosphorylation, Fc-fused ephrin-Fc proteins (5 µg) were clustered by incubation with 10 µg goat anti-human immunoglobulin G (IgG) (Jackson ImmunoResearch Inc., West Grove, PA, U.S.A., Cat# 109-005-008, RRID: AB_2337534) for 30 min at room temperature as previously described.^23,24) To inhibit the endocytosis of cell surface-bound proteins, the cells were incubated with Hanks’ balanced salt solution containing 0.1% sodium azide and 0.5 mg/mL bovine serum albumin for 30 min. The cells were then incubated with the recombinant protein for 30 min and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. The fixed cells were permeabilized with PBS containing 0.1% Triton X-100, and incubated with primary antibodies for 2 h. After three washes with PBS, the cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 for 2 h. Nuclei were stained with Hoechst33342 (2 µg/mL; Thermo Fisher Scientific). Fluorescent images were captured using BZ-X710 (Keyence, Osaka, Japan) or LSM 800 (Carl Zeiss, Jena, Germany).

Antibodies

Primary antibodies used in this study were as follows: goat polyclonal anti-Reelin (R&D Systems, Minneapolis, MN, U.S.A., Cat# AF3820, RRID:AB_2253745), rat monoclonal anti-Dab1 4H11 (MBL International, Schaumburg, IL, U.S.A., D354-3),²⁵⁾ goat polyclonal anti-EphA4 (R&D Systems Cat# AF641, RRID:AB_2099371), rabbit polyclonal anti-DDDDK-tag (MBL International, Cat# PM020, RRID:AB_591224), rat monoclonal anti-HA-tag (Roche, Basel, Switzerland, Cat# 11867423001, RRID:AB_390918), mouse monoclonal anti-phosphotyrosine (Millipore, Bedford, MA, U.S.A., Cat# 05-321, RRID:AB_309678), and rabbit polyclonal anti-GFP (MBL International, Cat# 598, RRID:AB_591819). Secondary antibodies used in this study were as follows: horseradish peroxidase (HRP)-linked anti-mouse IgG (Cytiva, Marlborough, MA, U.S.A., Cat# NA9310-1 mL, RRID:AB_772193), anti-rat IgG (Cytiva, Cat# NA935, RRID:AB_772207), or rabbit IgG (Cytiva, Cat# NA9340-1 mL, RRID:AB_772191), donkey Alexa488-conjugated anti-rabbit IgG (Thermo Fisher Scientific, Cat# A-21206, RRID:AB_2535792), goat Alexa488-conjugated anti-human IgG (Thermo Fisher Scientific, Cat# A-11013, RRID:AB_2534080), donkey Alexa594-conjugated anti-goat IgG (Thermo Fisher Scientific, Cat# A-11058, RRID:AB_2534105), and donkey Alexa594-conjugated anti-rat IgG (Thermo Fisher Scientific, Cat# A-21209, RRID:AB_2535795).

Statistical Analysis

All quantitative data are expressed as mean ± standard error, and individual data points are plotted. One-way ANOVA followed by Tukey’s multiple comparison test was used for multiple comparisons. Differences were considered significant at a p-value of <0.05. Statistical analyses were performed using Prism software (GraphPad Software, San Diego, CA, U.S.A., RRID: SCR_002798).

RESULTS

EphA4 Induces Tyrosine Phosphorylation in Dab1

To elucidate the non-canonical Dab1 phosphorylation pathway, we screened molecules that could induce tyrosine phosphorylation in Dab1 when co-expressed in COS-7 cells. We focused on membrane proteins that have previously been reported to interact with Reelin^18,26–30) or their family proteins. Candidate membrane proteins were co-expressed with HA-tagged Dab1 (Dab1-HA), and the phosphorylation of Dab1 was examined by Western blotting using anti-phosphotyrosine antibody 4G10. We found that the expression of VLDLR and ApoER2, the canonical Reelin receptors,^26,30) slightly increased phosphorylated Dab1 levels compared to that of the control (Fig. 1A, lanes 1–3). In contrast, the expression of Nrp1, APP, and ephrin-B1, membrane proteins recently reported to bind Reelin,^18,27,29) had no effect on the phosphorylated Dab1 level (Fig. 1A, lanes 4–6). Dab1 phosphorylation by EphB2 has been previously reported in HEK293T cells,²⁸⁾ and we confirmed EphB2-mediated Dab1 phosphorylation in our experimental system (Fig. 1A, lane 7). We found that both EphA4 and EphA3 could induce Dab1 phosphorylation and that EphA4 was more potent than EphA3 in Dab1 phosphorylation (Fig. 1A, lanes 8, 9). To better elucidate the differences in the efficiency of Dab1 phosphorylation between EphA4 and EphA3, the Dab1 protein was isolated by immunoprecipitation with an anti-HA antibody. EphA3 also induced Dab1 phosphorylation; however, its efficiency was markedly lower than that of EphA4 (Fig. 1B). However, EphA4 and EphA3 did not co-precipitate with Dab1 (Fig. 1B).

Fig. 1. EphA4 Induces Tyrosine Phosphorylation in Dab1

(A) EphB2, EphA4, and EphA3 induce Dab1 phosphorylation in COS-7 cells. The indicated transmembrane proteins were co-expressed with Dab1-HA in COS-7 cells. Cell lysates were prepared 24 h after transfection and analyzed by Western blotting using anti-phosphotyrosine (pY) and anti-HA antibodies. (B) EphA4 has a greater ability to phosphorylate Dab1 than EphA3. Dab1-HA was co-expressed with FLAG-EphA4 or FLAG-EphA3. pcDNA3.1Zeo+ was used as the empty vector. Cell lysates were prepared and immunoprecipitation with an anti-HA antibody was performed. Cell lysates (input) and precipitated proteins were analyzed by Western blotting using anti-pY, anti-FLAG, and anti-HA antibodies. Positions of the molecular mass markers (kDa) are shown on the left.

EphA4 Phosphorylates Dab1 via SFKs Activity

Reelin has been reported to directly bind to EphB2, which induces EphB2 phosphorylation.²⁸⁾ Therefore, we examined whether Reelin binds to EphA4. Recombinant Reelin was prepared and treated on COS-7 cells expressing FLAG-EphA4 or VLDLR-GFP. We confirmed Reelin binding to VLDLR (Figs. 2B, B’); however, we could not detect any interaction between Reelin and EphA4 (Figs. 2A, A’). Therefore, EphA4, in contrast to EphB2, is possibly not a Reelin receptor.

Fig. 2. Kinase Activity of EphA4 Is Required for Phosphorylation of Five Tyrosine Residues in Dab1

(A–B’) Immunostaining of cell surface-bound Reelin on COS-7 cells expressing FLAG-EphA4 (A, A’) or VLDLR-GFP (B, B’). Fixed cells were stained with anti-Reelin (A, B) and either anti-FLAG (A’) or anti-GFP (B’) antibodies. Scale bar: 50 µm. (C) The kinase activity of EphA4 is essential for Dab1 phosphorylation. The indicated expression plasmids were transfected into COS-7 cells, and the cell lysates were analyzed by Western Blotting using anti-pY, anti-FLAG, and anti-HA antibodies. (D) SFKs activity is required for EphA4-mediated Dab1 phosphorylation. The indicated expression plasmids were transfected into COS-7 cells. Cells were incubated with PP2 or PP3 for 5 h and cell lysates were analyzed by Western Blotting with anti-pY, anti-FLAG, and anti-HA antibodies. (E) Tyrosine residues of Dab1, which are phosphorylated by Reelin, are also phosphorylated by EphA4. The indicated expression plasmids were transfected into COS-7 cells, and the cell lysates were analyzed by Western Blotting using anti-pY, anti-FLAG, and anti-HA antibodies. Positions of the molecular mass markers (kDa) are shown on the left in C, D, and E

Two tyrosine residues in the cytoplasmic domain of EphA4 are important for its activation.³¹⁾ To investigate whether the kinase activity of EphA4 was required for Dab1 phosphorylation, we prepared two EphA4 mutant expression plasmids: kinase-dead EphA4 (Lys653 replaced by Met; EphA4-KM) and non-phosphorylatable EphA4 (Tyr596 and Tyr602 replaced by Phe; EphA4-YF). Both were expressed with Dab1-HA in COS-7 cells. As previously reported, EphA4 overexpression in COS-7 cells resulted in autophosphorylation without ephrin ligand stimulation,³¹⁾ whereas neither EphA4-KM nor EphA4-YF was phosphorylated (Fig. 2C). Dab1 was phosphorylated when co-expressed with EphA4 but not with EphA4-KM or EphA4-YF (Fig. 2C). These results indicate that the kinase activity of EphA4 is required for Dab1 phosphorylation.

SFKs regulate Reelin-mediated Dab1 phosphorylation.^9,10) To investigate whether SFKs are required for EphA4-induced Dab1 phosphorylation, we treated PP2, a SFKs inhibitor, in EphA4 and Dab1 co-expressing COS-7 cells. PP2 almost completely abolished EphA4-mediated Dab1 phosphorylation (Fig. 2D), whereas PP3, an inactive analog of PP2, had no such effect (Fig. 2D). Using a Dab1 mutant in which five tyrosine residues were replaced with phenylalanine, we found that five tyrosine residues (Y185, Y198, Y200, Y220, and Y232) of Dab1 were involved in its EphA4-mediated phosphorylation, (Dab1–5F, Fig. 2E). These results indicate that EphA4 induces tyrosine phosphorylation in Dab1 via the activation of SFKs, which is similar to Reelin signaling.

EphA-Mediated Dab1 Phosphorylation Was Not Observed in Primary Cultured Cortical Neurons

Eph receptors are classified into EphA and EphB subfamilies. With a few exceptions, ephrin-As bind to EphAs and ephrin-Bs bind to EphBs, inducing the phosphorylation of Eph receptors, for example, ephrin-A5 and ephrin-B1 have relatively high affinity for EphAs and EphBs, respectively.^16,32) To investigate the types of Ephs expressed on the cell surface of cultured cortical neurons, control-Fc, ephrin-A5-Fc, and ephrin-B1-Fc were prepared (Fig. 3A), and cultured cortical neurons were treated with clustered Fc proteins (Fig. 3B). We found that the puncta signals of ephrin-A5-Fc, but not ephrin-B1-Fc, were present on the cell surface of the neurons, suggesting that the cultured neurons mainly expressed EphAs, whereas the expression of EphBs was very low (Fig. 3B). We also found that Dab1 was expressed in both the soma and neurites of the cultured neurons (Fig. 3B).

Fig. 3. Cultured Cortical Neurons Have an Affinity for Ephrin-A5, but Not Ephrin-B1

(A) Western blot analysis of the purified Fc-fused proteins. Purified Fc-fused proteins were analyzed by Western blotting using an HRP-conjugated anti-human antibody. Positions of the molecular mass markers (kDa) are shown on the left. (B) Ephrin-A5-Fc, but not ephrin-B1-Fc, binds to primary cultured cortical neurons. Cortical neurons were incubated with clustered control-Fc, ephrin-A5-Fc, or ephrin-B1-Fc for 30 min. Cell surface-bound Fc proteins were detected using an Alexa Fluor 488-conjugated anti-human antibody (green), and fixed cells were immunostained with anti-Dab1 antibody (red). The nuclei were stained with Hoechst33342 (blue). Scale bar: 50 µm.

We stimulated primary cultured cortical neurons with different concentrations of clustered ephrin-A5-Fc. At all the concentrations tested, no increase was observed in Dab1 phosphorylation by ephrin-A5-Fc treatment (Fig. 4A). In our used culture system, some Reelin-producing cells are present.^33,34) Dab1 phosphorylation is induced by this endogenous Reelin and then the phosphorylated Dab1 is degraded,³⁵⁾ possibly making it difficult to detect the effects of ephrin-A5-Fc. Therefore, we prepared primary cultured cortical neurons from Reelin-deficient mice and incubated them with clustered ephrin-A5-Fc. EphA4 or Dab1 proteins were isolated by immunoprecipitation using anti-EphA4 or anti-Dab1 antibodies, and their phosphorylation was examined by Western blotting (Fig. 4B). We confirmed that ephrin-A5-Fc induced robust EphA4 phosphorylation (Fig. 4B, lane 2). Reelin also induced Dab1 phosphorylation (Fig. 4B, lane 3). However, ephrin-A5-Fc did not induce Dab1 phosphorylation (Figs. 4B, C). These results suggest that EphA4-mediated Dab1 phosphorylation rarely occurs in culture systems.

Fig. 4. EphA4 Activation Does Not Induce Dab1 Phosphorylation in the Cultured Cortical Neurons

(A) Ephrin-A5-Fc stimulation did not induce Dab1 phosphorylation. Primary cultured cortical neurons were incubated with different concentrations of clustered control-Fc (con) or ephrin-A5-Fc (A5), and cell lysates were subjected to Western blotting using or anti-Dab1 antibodies. Positions of the molecular mass markers (kDa) are shown on the left. (B) Ephrin-A5-Fc stimulation induced the phosphorylation of EphA4, but not Dab1, in primary cultured cortical neurons derived from Reelin-deficient mice. Cortical neurons from Reelin-deficient mice were incubated with control-Fc (lane 1), ephrin-A5-Fc (lane 2), or Reelin (lane 3) for 30 min. The cell lysates were subjected to immunoprecipitation with anti-Dab1 or anti-EphA4 antibodies, followed by Western blotting with anti-pY, anti-Dab1, or anti-EphA4 antibodies. Positions of the molecular mass markers (kDa) are shown on the left. (C) Quantitative analysis of phosphorylated Dab1. One-way ANOVA (F_{2, 8} = 8.408, p = 0.0182) followed by Tukey’s multiple comparison test was used to test for statistically significant differences. n = 3 experiments. NS, not significant.

DISCUSSION

Here, we show that EphA4 is a novel membrane protein that induces Dab1 phosphorylation. Biochemical screening of various membrane proteins revealed that EphA4 and EphA3 can induce Dab1 phosphorylation through SFKs (Figs. 1, 2). Phosphorylated EphA4 and EphA3 bind to SFKs, including Fyn,^36,37) suggesting that EphAs activate SFKs, which then phosphorylate Dab1. Interestingly, the ability of EphA3 to induce Dab1 phosphorylation was markedly lower than that of EphA4 (Fig. 1B). Because the homology between the cytoplasmic regions of EphA4 and EphA3 is relatively low,³⁸⁾ the efficiency of SFKs recruitment may differ between them. EphB2 has been reported to phosphorylate Dab1²⁸⁾; however, its biochemical properties differ considerably from those of EphA4. Reelin binds to EphB2 and induces its phosphorylation.²⁸⁾ However, Reelin did not bind to EphA4 (Fig. 2A) and EphA4 phosphorylation was not induced by Reelin stimulation in cultured cortical neurons (Fig. 4B). In addition, EphB2 binds to Dab1 via the DPXX sequence in the intracellular region,²⁸⁾ whereas EphA4 does not possess this motif.³⁹⁾ Indeed, EphA4 and Dab1 did not form a complex in overexpressed COS-7 cells (Fig. 1B). These findings suggest that Dab1 phosphorylation by EphA and EphB occurs through distinct mechanisms.

In cultured cortical neurons, EphA4 phosphorylation was induced by ephrin-A5-Fc stimulation, whereas Dab1 phosphorylation was undetectable (Fig. 4), suggesting that EphA4-mediated Dab1 phosphorylation only occurs under limited conditions. Dab1 and EphA4 may possibly exist in close proximity in overexpressed COS-7 cells, whereas they are segregated in different compartments in neurons. In the Reelin signaling pathway, Dab1 binds to the cytoplasmic region of ApoER2 or VLDLR,²⁶⁾ and the clustering of these receptors induced by multimerized Reelin promotes Dab1 phosphorylation.^40,41) One plausible model is that clustered Reelin receptors induce recruitment of Dab1 and SFKs, leading to efficient Dab1 phosphorylation.¹¹⁾ EphA4 did not form a complex with Dab1 (Fig. 1B), suggesting that EphA4 does not provide a scaffold for Dab1 in cultured neurons. EphA4 upregulation in neurons and glial cells has been reported in several conditions such as spinal cord injury,^42,43) propofol administration,⁴⁴⁾ and prenatal alcohol exposure.⁴⁵⁾ In addition, phosphorylated EphA4 is elevated in the brains of patients with depression or bipolar disorder.⁴⁶⁾ These findings suggest that EphA4-mediated Dab1 phosphorylation may occur under pathological conditions. Although our results were obtained from experiments using a cultured cell line, the next challenge is to elucidate the conditions under which EphA4 contributes to Dab1 phosphorylation in various cell types of the central nervous system.

In conclusion, we have identified a novel mechanism for EphA4-mediated Dab1 phosphorylation. Our findings provide clues for elucidating the mechanisms underlying Dab1 phosphorylation and Dab1-related pathologies.

Acknowledgments

We thank Profs. Tom Curran, John Flanagan, Kazunori Nakajima, and Junichi Takagi for generously providing expression plasmids.

Funding

This work was supported by JSPS KAKENHI Grant Numbers: JP20K07051 and 23K06119 (T.K.), and JP20H03384 (M.H.).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Howell BW, Hawkes R, Soriano P, Cooper JA. Neuronal position in the developing brain is regulated by mouse disabled-1. Nature, 389, 733–737 (1997).
• 2) Howell BW, Herrick TM, Hildebrand JD, Zhang Y, Cooper JA. Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development. Curr. Biol., 10, 877–885 (2000).
• 3) Sheldon M, Rice DS, D’Arcangelo G, Yoneshima H, Nakajima K, Mikoshiba K, Howell BW, Cooper JA, Goldowitz D, Curran T. Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice. Nature, 389, 730–733 (1997).
• 4) Yoneshima H, Nagata E, Matsumoto M, Yamada M, Nakajima K, Miyata T, Ogawa M, Mikoshiba K. A novel neurological mutant mouse, yotari, which exhibits reeler-like phenotype but expresses CR-50 antigen/Reelin. Neurosci. Res., 29, 217–223 (1997).
• 5) Yamamoto T, Setsu T, Okuyama-Yamamoto A, Terashima T. Histological study in the brain of the Reelin/Dab1-compound mutant mouse. Anat. Sci. Int., 84, 200–209 (2009).
• 6) Honda T, Hirota Y, Nakajima K. Heterozygous Dab1 null mutation disrupts neocortical and hippocampal development. eNeuro, 10, ENEURO.0433-22.2023 (2023).
• 7) Imai H, Shoji H, Ogata M, Kagawa Y, Owada Y, Miyakawa T, Sakimura K, Terashima T, Katsuyama Y. Dorsal forebrain-specific deficiency of Reelin-Dab1 signal causes behavioral abnormalities related to psychiatric disorders. Cereb. Cortex, 27, 3485–3501 (2017).
• 8) Smits DJ, Schot R, Wilke M, Van Slegtenhorst M, De Wit MCY, Dremmen MHG, Dobyns WB, Barkovich AJ, Mancini GMS. Biallelic DAB1 variants are associated with mild lissencephaly and cerebellar hypoplasia. Neurol. Genet., 7, e558 (2021).
• 9) Arnaud L, Ballif BA, Förster E, Cooper JA. Fyn tyrosine kinase is a critical regulator of Disabled-1 during brain development. Curr. Biol., 13, 9–17 (2003).
• 10) Bock HH, Herz J. Reelin activates SRC family tyrosine kinases in neurons. Curr. Biol., 13, 18–26 (2003).
• 11) Bock HH, May P. Canonical and non-canonical Reelin signaling. Front. Cell. Neurosci., 10, 166 (2016).
• 12) Keshvara L, Benhayon D, Magdaleno S, Curran T. Identification of Reelin-induced sites of tyrosyl phosphorylation on disabled 1. J. Biol. Chem., 276, 16008–16014 (2001).
• 13) Zhang JH, Zhao YF, He XX, Zhao Y, He ZX, Zhang L, Huang Y, Wang YB, Hu L, Liu L, Yu HL, Xu JH, Lai MM, Zhao DD, Cui L, Guo WX, Xiong WC, Ding YQ, Zhu XJ. DCC-mediated Dab1 phosphorylation participates in the multipolar-to-bipolar transition of migrating neurons. Cell Rep., 22, 3598–3611 (2018).
• 14) Dlugosz P, Teufl M, Schwab M, Kohl KE, Nimpf J. Disabled 1 is part of a signaling pathway activated by epidermal growth factor receptor. Int. J. Mol. Sci., 22, 1745 (2021).
• 15) Segarra M, Aburto MR, Cop F, Llaó-Cid C, Härtl R, Damm M, Bethani I, Parrilla M, Husainie D, Schänzer A, Schlierbach H, Acker T, Mohr L, Torres-Masjoan L, Ritter M, Acker-Palmer A. Endothelial Dab1 signaling orchestrates neuro-glia-vessel communication in the central nervous system. Science, 361, eaao2861 (2018).
• 16) Kania A, Klein R. Mechanisms of ephrin-Eph signalling in development, physiology and disease. Nat. Rev. Mol. Cell Biol., 17, 240–256 (2016).
• 17) Hibi T, Mizutani M, Baba A, Hattori M. Splicing variations in the ligand-binding domain of ApoER2 results in functional differences in the binding properties to Reelin. Neurosci. Res., 63, 251–258 (2009).
• 18) Kohno T, Ishii K, Hirota Y, Honda T, Makino M, Kawasaki T, Nakajima K, Hattori M. Reelin-Nrp1 interaction regulates neocortical dendrite development in a context-specific manner. J. Neurosci., 40, 8248–8261 (2020).
• 19) Sawahata M, Asano H, Nagai T, Ito N, Kohno T, Nabeshima T, Hattori M, Yamada K. Microinjection of Reelin into the mPFC prevents MK-801-induced recognition memory impairment in mice. Pharmacol. Res., 173, 105832 (2021).
• 20) Nakano Y, Kohno T, Hibi T, Kohno S, Baba A, Mikoshiba K, Nakajima K, Hattori M. The extremely conserved C-terminal region of Reelin is not necessary for secretion but is required for efficient activation of downstream signaling. J. Biol. Chem., 282, 20544–20552 (2007).
• 21) Kohno T, Honda T, Kubo KI, Nakano Y, Tsuchiya A, Murakami T, Banno H, Nakajima K, Hattori M. Importance of Reelin C-terminal region in the development and maintenance of the postnatal cerebral cortex and its regulation by specific proteolysis. J. Neurosci., 35, 4776–4787 (2015).
• 22) Kohno T, Ogino H, Yamakage Y, Hattori M. Expression and preparation of recombinant Reelin and ADAMTS-3 proteins. Methods Mol. Biol., 2043, 93–104 (2020).
• 23) Fu WY, Chen Y, Sahin M, Zhao XS, Shi L, Bikoff JB, Lai KO, Yung WH, Fu AKY, Greenberg ME, Ip NY. Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism. Nat. Neurosci., 10, 67–76 (2007).
• 24) Lawrenson ID, Wimmer-Kleikamp SH, Lock P, Schoenwaelder SM, Down M, Boyd AW, Alewood PF, Lackmann M. Ephrin-A5 induces rounding, blebbing and de-adhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling. J. Cell Sci., 115, 1059–1072 (2002).
• 25) Onoue A, Takeuchi M, Kohno T, Hattori M. Aberrant fragment of Dab1 protein is present in yotari mouse. Neurosci. Res., 88, 23–27 (2014).
• 26) Trommsdorff M, Gotthardt M, Hiesberger T, Shelton J, Stockinger W, Nimpf J, Hammer RE, Richardson JA, Herz J. Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2. Cell, 97, 689–701 (1999).
• 27) Hoe HS, Kea JL, Carney RSE, Lee J, Markova A, Lee JY, Howell BW, Hyman BT, Pak DTS, Bu G, Rebeck GW. Interaction of Reelin with amyloid precursor protein promotes neurite outgrowth. J. Neurosci., 29, 7459–7473 (2009).
• 28) Bouché E, Romero-Ortega MI, Henkemeyer M, Catchpole T, Leemhuis J, Frotscher M, May P, Herz J, Bock HH. Reelin induces EphB activation. Cell Res., 23, 473–490 (2013).
• 29) Sentürk A, Pfennig S, Weiss A, Burk K, Acker-Palmer A. Ephrin Bs are essential components of the Reelin pathway to regulate neuronal migration. Nature, 472, 356–360 (2011).
• 30) Hiesberger T, Trommsdorff M, Howell BW, Goffinet A, Mumby MC, Cooper JA, Herz J. Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of Disabled-1 and modulates tau phosphorylation. Neuron, 24, 481–489 (1999).
• 31) Kullander K, Mather NK, Diella F, Dottori M, Boyd AW, Klein R. Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo. Neuron, 29, 73–84 (2001).
• 32) Noberini R, Rubio De La Torre E, Pasquale EB. Profiling Eph receptor expression in cells and tissues: a targeted mass spectrometry approach. Cell Adh. Migr., 6, 102–156 (2012).
• 33) Kohno S, Kohno T, Nakano Y, Suzuki K, Ishii M, Tagami H, Baba A, Hattori M. Mechanism and significance of specific proteolytic cleavage of Reelin. Biochem. Biophys. Res. Commun., 380, 93–97 (2009).
• 34) Koie M, Okumura K, Hisanaga A, Kamei T, Sasaki K, Deng M, Baba A, Kohno T, Hattori M. Cleavage within Reelin repeat 3 regulates the duration and range of the signaling activity of Reelin protein. J. Biol. Chem., 289, 12922–12930 (2014).
• 35) Howell BW, Herrick TM, Cooper JA. Reelin-induced tyrosine phosphorylation of disabled 1 during neuronal positioning. Genes Dev., 13, 643–648 (1999).
• 36) Knöll B, Drescher U. Src family kinases are involved in EphA receptor-mediated retinal axon guidance. J. Neurosci., 24, 6248–6257 (2004).
• 37) Ellis C, Kasmi F, Ganju P, Walls E, Panayotou G, Reith AD. A juxtamembrane autophosphorylation site in the Eph family receptor tyrosine kinase, Sek, mediates high affinity interaction with p59fyn. Oncogene, 12, 1727–1736 (1996).
• 38) Wang Y, Shang Y, Li J, Chen W, Li G, Wan J, Liu W, Zhang M. Specific Eph receptor-cytoplasmic effector signaling mediated by SAM–SAM domain interactions. eLife, 7, 1–21 (2018).
• 39) Wybenga-Groot LE, Baskin B, Ong SH, Tong J, Pawson T, Sicheri F. Structural basis for autoinhibition of the EphB2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region. Cell, 106, 745–757 (2001).
• 40) Strasser V, Fasching D, Hauser C, Mayer H, Bock HH, Hiesberger T, Herz J, Weeber EJ, Sweatt JD, Pramatarova A, Howell B, Schneider WJ, Nimpf J. Receptor clustering is involved in Reelin signaling. Mol. Cell. Biol., 24, 1378–1386 (2004).
• 41) Yasui N, Kitago Y, Beppu A, Kohno T, Morishita S, Gomi H, Nagae M, Hattori M, Takagi J. Functional importance of covalent homodimer of Reelin protein linked via its central region. J. Biol. Chem., 286, 35247–35256 (2011).
• 42) Willson CA, Irizarry-Ramírez M, Gaskins HE, Cruz-Orengo L, Figueroa JD, Whittemore SR, Miranda JD. Upregulation of EphA receptor expression in the injured adult rat spinal cord. Cell Transplant., 11, 229–239 (2002).
• 43) Goldshmit Y, Galea MP, Wise G, Bartlett PF, Turnley AM. Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice. J. Neurosci., 24, 10064–10073 (2004).
• 44) Zhu X, Li H, Tian M, Zhou S, He Y, Zhou M. miR-455-3p alleviates propofol-induced neurotoxicity by reducing EphA4 expression in developing neurons. Biomarkers, 25, 685–692 (2020).
• 45) Altounian M, Bellon A, Mann F. Neuronal miR-17-5p contributes to interhemispheric cortical connectivity defects induced by prenatal alcohol exposure. Cell Rep., 42, 113020 (2023).
• 46) Zhang JC, Yao W, Qu Y, Nakamura M, Dong C, Yang C, Ren Q, Ma M, Han M, Shirayama Y, Hayashi-Takagi A, Hashimoto K. Increased EphA4-ephexin1 signaling in the medial prefrontal cortex plays a role in depression-like phenotype. Sci. Rep., 7, 7133 (2017).