Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.     

Regulation of protein tyrosine kinase signaling by substrate degradation during brain development

Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.     

Fyn tyrosine kinase is a critical regulator of disabled-1 during brain development

BACKGROUND: Disabled-1 (Dab1) is an intracellular adaptor protein that regulates migrations of various classes of neurons during mammalian brain development. Dab1 function depends on its tyrosine phosphorylation, which is stimulated by Reelin, an extracellular signaling molecule. Reelin increases the stoichiometry of Dab1 phosphorylation and downregulates Dab1 protein levels. Reelin binds to various cell surface receptors, including two members of the low-density lipoprotein receptor family that also bind to Dab1. Mutations in Dab1, its phosphorylation sites, Reelin, or the Reelin receptors cause a common phenotype. However, the molecular mechanism whereby Reelin regulates Dab1 tyrosine phosphorylation is poorly understood.RESULTS: We found that Reelin-induced Dab1 tyrosine phosphorylation in neuron cultures is inhibited by acute treatment with pharmacological inhibitors of Src family, but not Abl family, kinases. In addition, Reelin stimulates Src family kinases by a mechanism involving Dab1. We analyzed the Dab1 protein level and tyrosine phosphorylation stoichiometry by using brain samples and cultured neurons that were obtained from mouse embryos carrying mutations in Src family tyrosine kinases. We found that fyn is required for proper Dab1 levels and phosphorylation in vivo and in vitro. When fyn copy number is reduced, src, but not yes, becomes important, reflecting a partial redundancy between fyn and src.CONCLUSIONS: Reelin activates Fyn to phosphorylate and downregulate Dab1 during brain development. The results were unexpected because Fyn deficiency does not cause the same developmental phenotype as Dab1 or Reelin deficiency. This suggests additional complexity in the Reelin signaling pathway.     

Reelin activates SRC family tyrosine kinases in neurons

BACKGROUND: Reelin is a large signaling molecule that regulates the positioning of neurons in the mammalian brain. Transmission of the Reelin signal to migrating embryonic neurons requires binding to the very-low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor-2 (apoER2). This induces tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1), which interacts with a shared sequence motif in the cytoplasmic tails of both receptors. However, the kinases that mediate Dab1 tyrosine phosphorylation and the intracellular pathways that are triggered by this event remain unknown. RESULTS: We show that Reelin activates members of the Src family of non-receptor tyrosine kinases (SFKs) and that this activation is dependent on the Reelin receptors apoER2 and VLDLR and the adaptor protein Dab1. Dab1 is tyrosine phosphorylated by SFKs, and the kinases themselves can be further activated by phosphorylated Dab1. Increased Dab1 protein expression in fyn-deficient mice implies a response to impaired Reelin signaling that is also observed in mice lacking Reelin or its receptors. However, fyn deficiency alone does not compound the neuronal positioning defect of vldlr- or apoer2-deficient mice, and this finding suggests functional compensation by other SFKs. CONCLUSIONS: Our results show that Dab1 is a physiological substrate as well as an activator of SFKs in neurons. Based on genetic evidence gained from multiple strains of mutant mice with defects in Reelin signaling, we conclude that activation of SFKs is a normal part of the cellular Reelin response.     

Reelin binds alpha3beta1 integrin and inhibits neuronal migration

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.     

Apolipoprotein E receptors are required for reelin-induced proteasomal degradation of the neuronal adaptor protein Disabled-1

The cytoplasmic adaptor protein Disabled-1 (Dab1) is necessary for the regulation of neuronal positioning in the developing brain by the secreted molecule Reelin. Binding of Reelin to the neuronal apolipoprotein E receptors apoER2 and very low density lipoprotein receptor induces tyrosine phosphorylation of Dab1 and the subsequent activation or relocalization of downstream targets like phosphatidylinositol 3 (PI3)-kinase and Nckbeta. Disruption of Reelin signaling leads to the accumulation of Dab1 protein in the brains of genetically modified mice, suggesting that Reelin limits its own action in responsive neurons by down-regulating the levels of Dab1 expression. Here, we use cultured primary embryonic neurons as a model to demonstrate that Reelin treatment targets Dab1 for proteolytic degradation by the ubiquitin-proteasome pathway. We show that tyrosine phosphorylation of Dab1 but not PI3-kinase activation is required for its proteasomal targeting. Genetic deficiency in the Dab1 kinase Fyn prevents Dab1 degradation. The Reelin-induced Dab1 degradation also depends on apoER2 and very low density lipoprotein receptor in a gene-dose dependent manner. Moreover, pharmacological blockade of the proteasome prevents the formation of a proper cortical plate in an in vitro slice culture assay. Our results demonstrate that signaling through neuronal apoE receptors can activate the ubiquitin-proteasome machinery, which might have implications for the role of Reelin during neurodevelopment and in the regulation of synaptic transmission.     

Reconstitution of the Reelin signaling pathway in fibroblasts demonstrates that Dab1 phosphorylation is independent of receptor localization in lipid rafts

The Reelin signaling pathway operates in migrating neurons and is indispensable for their correct positioning during embryonic brain development. Many biochemical and cell biological studies to dissect the Reelin pathway at the molecular level are hampered by the lack of a cell line harboring a functional Reelin signaling pathway. Here we present fibroblast cell lines in which all required functional components of the pathway have been reconstituted. These cells react upon Reelin treatment in the same way as primary neurons. We have subsequently used these cell lines to study the subcellular localization of ApoER2 and the VLDL receptor and could demonstrate that receptor-mediated Dab1 phosphorylation does not depend on lipid rafts and that phosphorylated Dab1 remains bound to the receptor tail when the pathway is activated by Reelin.     

Reelin promotes hippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway

Reelin is a secreted glycoprotein that regulates neuronal positioning in cortical brain structures through the VLDLR and ApoER2 receptors and the adaptor protein Dab1. In addition to cellular disorganization, dendrite abnormalities are present in the brain of reeler mice lacking Reelin. It is unclear whether these defects are due primarily to cellular ectopia or the absence of Reelin. Here we examined dendrite development in the hippocampus of normal and mutant mice and in dissociated cultures. We found that dendrite complexity is severely reduced in homozygous mice deficient in Reelin signaling both in vivo and in vitro, and it is also reduced in heterozygous mice in the absence of cellular ectopia. Addition of Reelin interfering antibodies, receptor antagonists, and Dab1 phosphorylation inhibitors prevented dendrite outgrowth from normal neurons, whereas addition of recombinant Reelin rescued the deficit in reeler cultures. Thus, the same signaling pathway controls both neuronal migration and dendrite maturation.     

Regulation of Cortical Neuron Migration by the Reelin Signaling Pathway

Reeler is a mutant mouse with defects in layered structures of the central nervous system, such as the cerebral cortex, hippocampus, and cerebellum, and has been extensively examined for more than half a century. The full-length cDNA for the responsible gene for reeler, reelin, was serendipitously identified, revealing that Reelin encodes a large secreted protein. So far, two Reelin receptors, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, and the cytoplasmic adaptor protein Disabled homolog 1 (Dab1) have been shown to be essential for Reelin signaling. Although a number of downstream cascades of Dab1 have also been reported using various experimental systems, the physiological functions of Reelin in vivo remain controversial. Here, we review recent advances in the understanding of the Reelin-Dab1 signaling pathway in the developing cerebral cortex.     

Serine phosphorylation regulates disabled-1 early isoform turnover independently of Reelin

The Reelin-Disabled 1 (Dab1) signaling pathway plays an important role in neuronal cell migration during brain development. Dab1, an intracellular adapter protein which is tyrosine phosphorylated upon Reelin stimulation, has been directly implicated in the transmission and termination of Reelin-mediated signaling. Two main forms of Dab1 have been identified in the developing chick retina, an early isoform (Dab1-E) expressed in progenitor cells and a late isoform (Dab1-L, a.k.a. Dab1) expressed in differentiated cells. Dab1-E is missing two Src family kinase (SFK) phosphorylation sites that are critical for Reelin-Dab1 signaling and is not tyrosine phosphorylated. We have recently demonstrated a role for Dab1-E in the maintenance of retinal progenitor cells. Here, we report that Dab1-E is phosphorylated at serine/threonine residues independent of Reelin. Cdk2, highly expressed in retinal progenitor cells, mediates Dab1-E phosphorylation at serine 475 which in turn promotes ubiquitination-triggered proteasome degradation of Dab1-E. Inhibition of protein phosphatase 1 and/or protein phosphatase 2A leads to increased Dab1-E instability. We propose that Dab1 turnover is regulated by both Reelin-independent serine/threonine phosphorylation and Reelin-dependent tyrosine phosphorylation.     

Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth

Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3beta (GSK3beta) are not. On the other hand, mTor, but not GSK3beta, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.     

Disabled1 regulates the intracellular trafficking of reelin receptors

Reelin is a huge secreted protein that controls proper laminar formation in the developing brain. It is generally believed that tyrosine phosphorylation of Disabled1 (Dab1) by Src family tyrosine kinases is the most critical downstream event in Reelin signaling. The receptors for Reelin belong to the low density lipoprotein receptor family, most of whose members undergo regulated intracellular trafficking. In this study, we propose novel roles for Dab1 in Reelin signaling. We first demonstrated that cell surface expression of Reelin receptors was decreased in Dab1-deficient neurons. In heterologous cells, Dab1 enhanced cell surface expression of Reelin receptors, and this effect was mediated by direct interaction with the receptors. Moreover, Dab1 did not stably associate with the receptors at the plasma membrane in the resting state. When Reelin was added to primary cortical neurons, Dab1 was recruited to the receptors, and its tyrosine residues were phosphorylated. Although Reelin and Dab1 colocalized well shortly after the addition of Reelin, Dab1 was no longer associated with internalized Reelin. When Src family tyrosine kinases were inhibited, internalization of Reelin was severely abrogated, and Reelin colocalized with Dab1 near the plasma membrane for a prolonged period. Taken together, these results indicate that Dab1 regulates both cell surface expression and internalization of Reelin receptors, and these regulations may play a role in correct laminar formation in the developing brain.     

Signaling through Disabled 1 requires phosphoinositide binding

The Reelin signaling pathway plays a critical role in the correct positioning of neurons within the developing brain. Within this pathway, Disabled 1 (Dab1) serves as an intracellular adaptor that is tyrosine phosphorylated when Reelin, a secreted glycoprotein, binds to the lipoprotein receptors VLDLR and ApoER2 on the surface of neurons. The phosphotyrosine-binding (PTB) domain within its amino terminus enables Dab1 to recognize and bind to a conserved sequence motif within the cytoplasmic tails of the receptors. In addition, the PTB contains a Pleckstrin Homology-like subdomain that binds to phosphoinositides. Here, we show that the phosphoinositide-binding region within Dab1 PTB domain is required for membrane localization and basal tyrosine phosphorylation of Dab1 independently of VLDLR and ApoER2. Furthermore, receptor-independent membrane targeting of Dab1 is required for its interaction with Src and Crk, and disruption of phosphoinositide binding also blocks subsequent Reelin-induced tyrosine phosphorylation of Dab1.     

Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and Reelin signal transduction

Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P2 through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.     

Dual functions of Dab1 during brain development

Reelin coordinates the movements of neurons during brain development by signaling through the Dab1 adaptor and Src family tyrosine kinases. Experiments with cultured neurons have shown that when Dab1 is phosphorylated on tyrosine, it activates Akt and provides a scaffold for assembling signaling complexes, including the paralogous Crk and CrkL adaptors. The roles of Akt and Dab1 complexes during development have been unclear. We have generated two Dab1 alleles, each lacking two out of the four putative tyrosine phosphorylation sites. Neither allele supports normal brain development, but each allele complements the other. Two tyrosines are required for Reelin to stimulate Dab1 phosphorylation at the other sites, to activate Akt, and to downregulate Dab1 levels. The other two tyrosines are required to stimulate a Crk/CrkL-C3G pathway. The absence of Crk/CrkL binding sites and C3G activation causes an unusual layering phenotype. These results show that Reelin-induced Akt stimulation and Dab1 turnover are not sufficient for normal development and suggest that Dab1 acts both as a kinase switch and as a scaffold for assembling signaling complexes in vivo.     

Mouse disabled 1 regulates the nuclear position of neurons in a Drosophila eye model

Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Here we show that Disabled 1 (Dab1), which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, we observed that a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhanced the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduced the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling which regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons.     

Tyrosine phosphorylated Disabled 1 recruits Crk family adapter proteins

Disabled 1 (Dab1) functions as a critical adapter protein in the Reelin signaling pathway to direct proper positioning of neurons during brain development. Reelin stimulates phosphorylation of Dab1 on tyrosines 198 and 220, and phosphorylated Dab1 is likely to interact with downstream signaling proteins that contain Src homology 2 (SH2) domains. To search for such proteins, we used a Sepharose-conjugated peptide containing phosphotyrosine 220 (PTyr-220) of Dab1, as an affinity matrix to capture binding proteins from mouse brain extracts. Mass spectrometric analysis of bound proteins revealed that Crk family adapter proteins selectively associated with this phosphorylation site. We further show that Crk-I and Crk-II, but not CrkL, stimulate phosphorylation of Dab1 on tyrosine 220 in a Src-dependent manner. Our results suggest that Crk family adapter proteins may play an important role in the Reelin signaling pathway during brain development.     

Reelin is a ligand for lipoprotein receptors

A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.     

Phosphatidylinositol 3-kinase interacts with the adaptor protein Dab1 in response to Reelin signaling and is required for normal cortical lamination

Reelin is a large secreted signaling protein that binds to two members of the low density lipoprotein receptor family, the apolipoprotein E receptor 2 and the very low density lipoprotein receptor, and regulates neuronal positioning during brain development. Reelin signaling requires activation of Src family kinases as well as tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This results in activation of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and the inhibition of glycogen synthase kinase 3beta, a protein that is implicated in the regulation of axonal transport. Here we demonstrate that PI3K activation by Reelin requires Src family kinase activity and depends on the Reelin-triggered interaction of Dab1 with the PI3K regulatory subunit p85alpha. Because the Dab1 phosphotyrosine binding domain can interact simultaneously with membrane lipids and with the intracellular domains of apolipoprotein E receptor 2 and very low density lipoprotein receptor, Dab1 is preferentially recruited to the neuronal plasma membrane, where it is phosphorylated. Efficient Dab1 phosphorylation and activation of the Reelin signaling cascade is impaired by cholesterol depletion of the plasma membrane. Using a neuronal migration assay, we also show that PI3K signaling is required for the formation of a normal cortical plate, a step that is dependent upon Reelin signaling.     

Disabled-1 interacts with a novel developmentally regulated protocadherin.

Disabled-1 (Dab1) is an intracellular adapter protein that mediates the effect of Reelin on neuronal migration and cell positioning during mammalian brain development. To identify components of the Reelin-Dab1 signaling pathway, we searched for proteins that interact with Dab1 using a yeast two-hybrid strategy. We found that the Dab1 phosphotyrosine binding (PTB) domain interacts with a novel protocadherin, orthologous to human protocadherin 18. Mouse Pcdh18 (mPcdh18), which consists of four exons similar to other protocadherin family members, maps to chromosome 3. The deduced amino acid sequence of mPcdh18 contains six extracellular cadherin motifs, a single transmembrane region, and a large intracellular domain. The site of Dab1 interaction was localized to the C-terminal 243 residues of mPcdh18. Expression analyses revealed that mPcdh18 is present in a variety of tissues in the embryo, but in adult mice it is primarily expressed in lung and kidney. In embryonic brain, mPcdh18 expression is temporally and spatially regulated. Our results indicate that mPcdh18 participates in signaling pathways involving PTB-containing proteins and suggest that it may play a role during brain development.