Tyrosine Phosphorylation Of Dab1 Research Articles

A key requirement for synaptic Reelin signaling in ketamine-mediated behavioral and synaptic action

Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that produces rapid antidepressant action in some patients with treatment-resistant depression. However, recent data suggest that ∼50% of patients with treatment-resistant depression do not respond to ketamine. The factors that contribute to the nonresponsiveness to ketamine's antidepressant action remain unclear. Recent studies have reported a role for secreted glycoprotein Reelin in regulating pre- and postsynaptic function, which suggests that Reelin may be involved in ketamine's antidepressant action, although the premise has not been tested. Here, we investigated whether the disruption of Reelin-mediated synaptic signaling alters ketamine-triggered synaptic plasticity and behavioral effects. To this end, we used mouse models with genetic deletion of Reelin or apolipoprotein E receptor 2 (Apoer2), as well as pharmacological inhibition of their downstream effectors, Src family kinases (SFKs) or phosphoinositide 3-kinase. We found that disruption of Reelin, Apoer2, or SFKs blocks ketamine-driven behavioral changes and synaptic plasticity in the hippocampal CA1 region. Although ketamine administration did not affect tyrosine phosphorylation of DAB1, an adaptor protein linked to downstream signaling of Reelin, disruption of Apoer2 or SFKs impaired baseline NMDA receptor-mediated neurotransmission. These results suggest that maintenance of baseline NMDA receptor function by Reelin signaling may be a key permissive factor required for ketamine's antidepressant effects. Taken together, our results suggest that impairments in Reelin-Apoer2-SFK pathway components may in part underlie nonresponsiveness to ketamine's antidepressant action.

RELN signaling modulates glioblastoma growth and substrate-dependent migration.

Glioblastoma (GBM) represents the most common and most malignant type of primary brain tumor and significantly contributes to cancer morbidity and mortality. Invasion into the healthy brain parenchyma is a major feature of glioblastoma aggressiveness. Reelin (RELN) is a large secreted extracellular matrix glycoprotein that regulates neuronal migration and positioning in the developing brain and sustains functionality in the adult brain. We here show that both RELN and its main downstream effector DAB1 are silenced in glioblastoma as compared to non-neoplastic tissue and mRNA expression is inversely correlated with malignancy grade. Furthermore, RELN expression is positively correlated with patient survival in two large, independent clinically annotated datasets. RELN silencing occurs via promoter hypermethylation as shown by both database mining and bisulfite sequencing of the RELN promoter. Consequently, treatment with 5'-Azacytidine and trichostatin A induced RELN expression in vitro. On the functional level, we found RELN to regulate glioblastoma cell migration both in a DAB1 (tyrosine phosphorylation)-dependent and -independent fashion, depending on the substrate provided. Moreover, stimulation of RELN signaling strongly reduced proliferation in glioblastoma cells. This phenotype depends on DAB1 stimulation by RELN, as a mutant that lacks all RELN induced tyrosine phosphorylation sites (DAB1-5F) failed to induce a growth arrest. Proteomic analyzes revealed that these effects are mediated by a reduction in E2F targets and dephosphorylation of ERK1/2. Taken together, our data establish a relevance of RELN signaling in glioblastoma pathology and thereby might unearth novel, yet unrecognized treatment options.

Mathematical model of early Reelin-induced Src family kinase-mediated signaling.

Reelin is a large glycoprotein with a dual role in the mammalian brain. It regulates the positioning and differentiation of postmitotic neurons during brain development and modulates neurotransmission and memory formation in the adult brain. Alterations in the Reelin signaling pathway have been described in different psychiatric disorders. Reelin mainly signals by binding to the lipoprotein receptors Vldlr and ApoER2, which induces tyrosine phosphorylation of the adaptor protein Dab1 mediated by Src family kinases (SFKs). In turn, phosphorylated Dab1 activates downstream signaling cascades, including PI3-kinase-dependent signaling. In this work, a mechanistic model based on ordinary differential equations was built to model early dynamics of the Reelin-mediated signaling cascade. Mechanistic models are frequently used to disentangle the highly complex mechanisms underlying cellular processes and obtain new biological insights. The model was calibrated on time-resolved data and a dose-response measurement of protein concentrations measured in cortical neurons treated with Reelin. It focusses on the interplay between Dab1 and SFKs with a special emphasis on the tyrosine phosphorylation of Dab1, and their role for the regulation of Reelin-induced signaling. Model selection was performed on different model structures and a comprehensive mechanistic model of the early Reelin signaling cascade is provided in this work. It emphasizes the importance of Reelin-induced lipoprotein receptor clustering for SFK-mediated Dab1 trans-phosphorylation and does not require co-receptors to describe the measured data. The model is freely available within the open-source framework Data2Dynamics (www.data2dynamics.org). It can be used to generate predictions that can be validated experimentally, and provides a platform for model extensions both to downstream targets such as transcription factors and interactions with other transmembrane proteins and neuronal signaling pathways.

Alternative Splicing of Disabled-1 Controls Multipolar-to-Bipolar Transition of Migrating Neurons in the Neocortex.

Multipolar-to-bipolar transition (MBT) is crucial for the neuronal migration and positioning in the neocortex. Reelin-Disabled-1 (Dab1) signaling plays a pivotal role in neuronal migration, yet how Dab1 coordinatively regulates downstream molecules to affect MBT remains unclear. We have previously found that alternative splicing produces multiple Dab1 isoforms with different tyrosine motifs and differential ability to recruit downstream effectors. Here, we report that splicing of Dab1 exons 7 and 8 and 9bc dynamically regulates the inclusion and activities of Dab1 tyrosine motifs in the neocortex. By in utero electroporation, we show that expression of Dab1 isoforms missing exons 7 and 8 or retaining exons 9bc in WT neurons resulted in neuronal migration defects with attenuated Dab1 tyrosine phosphorylation, disrupted leading process extension, and disorientated multipolar neurons in the multipolar accumulation zone. Introducing the canonical Dab1 form, but not those missing exons 7 and 8 or retaining exons 9bc, into Dab1-deficient neurons promoted MBT and rescued neuronal migration defects, suggesting that alternative splicing of Dab1 modulates the tyrosine motif switch and mediates MBT of cortical neurons. Our study reveals a critical mechanism by which Dab1 alternative splicing coordinately controls MBT and neuronal migration in a spatiotemporal manner.

Dab1 stabilizes its interaction with Cin85 by suppressing Cin85 phosphorylation at serine 587

Crk and CrkL adaptors play essential neuronal positioning roles downstream of Reelin-induced Dab1 tyrosine phosphorylation. Recently we identified Cin85 to be a CrkL-SH3 binding partner from embryonic murine brain while others found Cin85 binds directly to Dab1. Here using mass spectrometry, biochemical and mutational analyses we show that Dab1 suppresses Cin85 phosphorylation at Ser587. Furthermore a Cin85 Ser587 phosphomimetic disrupts the Dab1-Cin85 complex without affecting the Cin85-CapZ complex. These data provide an early glimpse into how Cin85 phosphorylation might alter the composition of its scaffolding partners to regulate its diverse roles including vesicular trafficking, receptor endocytosis and actin remodeling.

Reelin-Disabled-1 signaling in neuronal migration: splicing takes the stage

Reelin-Disabled-1 (Dab1) signaling has a well-established role in regulating neuronal migration during brain development. Binding of Reelin to its receptors induces Dab1 tyrosine phosphorylation. Tyrosine-phosphorylated Dab1 recruits a wide range of SH2 domain-containing proteins and activates multiple signaling cascades, resulting in cytoskeleton remodeling and precise neuronal positioning. In this review, we summarize recent progress in the Reelin-Dab1 signaling field. We focus on Dab1 alternative splicing as a mechanism for modulating the Reelin signal in developing brain. We suggest that correct positioning of neurons in the developing brain is at least partly controlled by alternatively-spliced Dab1 isoforms that differ in the number and type of tyrosine phosphorylation motifs that they contain. We propose a model whereby different subsets of SH2 domain-containing proteins are activated by different Dab1 isoforms, resulting in coordinated migration of neurons.

Splice-Mediated Motif Switching Regulates Disabled-1 Phosphorylation and SH2 Domain Interactions

Disabled-1 (Dab1) plays a key role in reelin-mediated neuronal migration during brain development. Tyrosine phosphorylation of Dab1 at two YQXI and two YXVP motifs recruits multiple SH2 domains, resulting in activation of a wide range of signaling cascades. However, the molecular mechanisms underlying the coordinated regulation of Dab1 downstream effectors remain poorly understood. Here, we show that alternative splicing results in inclusion of different combinations of YQXI and YXVP motifs in Dab1 isoforms during development. Dab1 variants with partial or complete loss of YQXI motifs are preferentially expressed at early developmental stages, whereas the commonly studied Dab1 is predominantly expressed at late developmental stages. Expression of Dab1 variants in 293T and Neuro2a cells reveals reduced levels or absence of tyrosine phosphorylation in variants that have lost one or both YQXI motifs. We further demonstrate that Dab1 variants differ in their abilities to activate Src and recruit distinct SH2 domains involved in specific downstream signaling pathways. We propose that coordinated expression of specific Dab1 isoforms in different populations of cells in the developing brain contributes to precise neuronal migration by modulating the activity of subsets of Dab1 downstream effectors.

Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.

Disabled-1 Alternative Splicing in Human Fetal Retina and Neural Tumors

BackgroundThe Reelin-Dab1 signaling pathway plays a critical role in the positioning of migrating neurons, dendrite formation and lamination in the developing central nervous system. We have previously identified two alternatively spliced forms of Dab1 in the developing chick retina: an early form, Dab1-E, expressed in retinal progenitor cells, and a late form, Dab1 or Dab1-L, expressed in amacrine and ganglion cells. Compared to Dab1-L, Dab1-E lacks two exons that encode two Src family kinase (SFK) phosphorylation sites.Principal FindingsBoth Dab1-L and Dab1-E-like transcripts were identified in human fetal retina. Expression of human Dab1-L in primary chick retinal cultures resulted in Reelin-mediated induction of SFK phosphorylation and formation of neurite-like processes. In contrast, human Dab1-E-expressing cells retained an undifferentiated morphology. The human Dab1 gene is located within a common fragile site, and it has been postulated that it may function as a tumor suppressor. Analysis of Dab1 splice forms in retinoblastoma and neuroblastoma tumor cells revealed relative enrichment of Dab1-L-like (includes exons 7 and 8) and Dab1-E-like (excludes exons 7 and 8) transcripts in retinoblastoma and neuroblastoma, respectively. Treatment of retinoblastoma cell line RB522A with Reelin resulted in increased tyrosine phosphorylation of Dab1. As Nova2 has previously been implicated in the exclusion of exons 9B and 9C in Dab1, we examined the expression of this splicing factor in neuroblastoma and retinoblastoma cell lines. Nova2 was only detected in neuroblastoma cells, suggesting a correlation between Nova2 expression and increased levels of Dab1-E-like splice forms in neuroblastoma.ConclusionsThese results indicate that alternative splicing of Dab1 is conserved in avian and mammalian species, with Dab1-L driving SFK phosphorylation in both species. Dab1-E- and Dab-L-like isoforms are also expressed in childhood neural tumors, with preferential enrichment of Dab1-L-like and Dab1-E-like isoforms in retinoblastoma and neuroblastoma, respectively.

Identification of CrkL-SH3 Binding Proteins from Embryonic Murine Brain: Implications for Reelin Signaling during Brain Development

The Crk and Crk-like (CrkL) adaptor proteins play important roles in numerous signaling pathways, bridging tyrosine kinase substrates to downstream signaling effectors by virtue of their phosphotyrosine-binding SH2 domains and their effector-binding SH3 domains. Critical to understanding the diverse roles of Crk/CrkL is the identification of tissue- and signal-specific tyrosine phosphorylated substrates to which they are recruited and the tissue-specific effector proteins they chaperone into signaling complexes. Crk and CrkL are known biochemically and genetically to be essential mediators of Reelin/Disabled-1 (Dab1) signaling, which governs proper mammalian brain development. Multimeric Reelin clusters its receptors as well as the receptor-bound intracellular scaffolding protein Dab1. Clustering induces Fyn/Src-dependent Dab1 tyrosine phosphorylation, which recruits Crk/CrkL and SH3-bound effectors. Previously, 21 Crk/CrkL-SH3 binding proteins were identified from diverse cell types. We present here the proteomic identification of 101 CrkL-SH3 binding proteins from embryonic murine brain. The identified proteins are enriched in the Crk/CrkL-SH3 binding motif and signaling activities regulating cell adhesion and motility. These results suggest Reelin-induced Dab1 tyrosine phosphorylation may generate a multifaceted signaling scaffold containing a rich array of Crk/CrkL-SH3 binding effectors and may explain a growing diversity of cellular activities suggested to be influenced by Reelin/Dab1 signaling.

Mechanism of cerebellar development as revealed by Reelin CTR-deficient mouse

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.

Crk and Crk-Like Play Essential Overlapping Roles Downstream of Disabled-1 in the Reelin Pathway

Reelin controls neuronal positioning in the developing brain by binding to the two lipoprotein receptors, very-low-density lipoprotein receptor and apolipoprotein E receptor 2, to stimulate phosphorylation of Disabled-1 (Dab1) by the Fyn and Src tyrosine kinases. Crk and Crk-like (CrkL) have been proposed to interact with tyrosine phosphorylated Dab1 to mediate downstream events in the Reelin pathway. However, these adaptor proteins are widely expressed, and they fulfill essential functions during embryonic development. To address their specific roles in Reelin-mediated neuronal migration, we generated mutant mice, by Cre-loxP recombination, lacking Crk and CrkL in most neurons. These animals displayed the major anatomic features of reeler including, cerebellar hypofoliation, failure of Purkinje cell migration, absence of preplate splitting, impaired dendritic development, and disruption of layer formation in the hippocampus and cerebral cortex. However, proximal signaling involving tyrosine phosphorylation and turnover of Dab1 occurred normally in the mutant mouse brain and in primary cortical neurons treated with Reelin. In contrast, two downstream signaling events, Reelin-induced phosphorylation of C3G and Akt, were not observed in the absence of Crk and CrkL in mouse embryonic cortical neurons. These findings place C3G and Akt phosphorylation downstream of Crk and CrkL, which play essential overlapping functions in the Reelin signaling pathway.

Fibrillar prion peptide PrP(106–126) treatment induces Dab1 phosphorylation and impairs APP processing and Aβ production in cortical neurons

Alzheimer’s disease and prion diseases (e.g., Creutzfeldt–Jakob disease) display profound neural lesions associated with aberrant protein processing and extracellular amyloid deposits. However, the intracellular events in prion diseases and their relation with the processing of the amyloid precursor protein (APP) and β-amyloid generation are unknown. The adaptor protein Dab1 may regulate intracellular trafficking and secretase-mediated proteolysis in APP processing. However, a putative relationship between prion diseases and Dab1/APP interactions is lacking. Thus, we examined, in inoculated animals, whether Dab1 and APP processing are targets of the intracellular events triggered by extracellular exposure to PrP(106–126) peptide. Our in vitro results indicate that PrP(106–126) peptide induces tyrosine phosphorylation of Dab1 by activated members of the Src family of tyrosine kinases (SFK), which implies further Dab1 degradation. We also corroborate these results in Dab1 protein levels in prion-inoculated hamsters. Finally, we show that fibrillar prion peptides have a dual effect on APP processing and β-amyloid production. First, they block APP trafficking at the cell membrane, thus decreasing β-amyloid production. In parallel, they reduce Dab1 levels, which also alter APP processing. Lastly, neuronal cultures from Dab1-deficient mice showed severe impairment of APP processing with reduced sAPP secretion and Aβ production after prion peptide incubation. Taken together, these data indicate a link between intracellular events induced by exposure to extracellular fibrillar peptide or PrP res, and APP processing and implicate Dab1 in this link.

PO4-100 CONJUGATED LINOLEIC ACID ALTERS THE PRO-ATHEROSCLEROTIC PHENOTYPE OF THE MONOCYTE/MACROPHAGE - EVIDENCE FOR THE INVOLVEMENT OF A NOVEL SIGNALLING PATHWAY

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.

Migration of sympathetic preganglionic neurons in the spinal cord is regulated by reelin‐dependent Dab1 tyrosine phosphorylation and CrkL

The actions of Reelin in neuronal positioning in the developing cortex and cerebellum are relayed by Src-family kinase (SFK)-mediated phosphorylation of Dab1. Biochemical studies show that after phosphorylation Dab1 binds to an adaptor protein, CrkL. Whether CrkL is important for Reelin signaling in vivo is unknown, because crkl(-/-) embryos die before cortical development is complete. In the developing spinal cord, Reelin and components of its signaling pathway, VLDLR, ApoER2, and Dab1, control the positioning of sympathetic preganglionic neurons (SPN); however, it is not known whether SFKs or Dab1 tyrosine phosphorylation is required. In the present study, we asked whether Reelin-controlled SPN migration depends on tyrosine phosphorylation of Dab1 by SFKs and whether CrkL is involved in SPN migration. To answer these questions, we examined the location of SPN in various mutant mouse embryos. Results showed that, in dab1(5F/5F) embryos, which express a nonphosphorylated mutant of Dab1, and in src(-/-)fyn(-/-) double knockout embryos, the location of SPN is identical to that of reeler. These results show that tyrosine phosphorylation of Dab1 by SFKs is required for Reelin-regulated SPN positioning. In addition, we found that SPN migration in crkl(-/-) showed a partial reeler phenotype, suggesting a partial loss of response of SPN to Reelin signaling. These results suggest a role for CrkL in the Reelin signaling pathway to control neuronal migration.

Hierarchical Disabled-1 Tyrosine Phosphorylation in Src family Kinase Activation and Neurite Formation

There are two developmentally regulated alternatively spliced forms of Disabled-1 (Dab1) in the chick retina: an early form (Dab1-E) expressed in retinal precursor cells and a late form (Dab1-L) expressed in neuronal cells. The main difference between these two isoforms is the absence of two Src family kinase (SFK) recognition sites in Dab1-E. Both forms retain two Abl/Crk/Nck recognition sites implicated in the recruitment of SH2 domain-containing signaling proteins. One of the Dab1-L-specific SFK recognition sites, at tyrosine(Y)-198, has been shown to be phosphorylated in Reelin-stimulated neurons. Here, we use Reelin-expressing primary retinal cultures to investigate the role of the four Dab1 tyrosine phosphorylation sites on overall tyrosine phosphorylation, Dab1 phosphorylation, SFK activation and neurite formation. We show that Y198 is essential but not sufficient for maximal Dab1 phosphorylation, SFK activation and neurite formation, with Y232 and Y220 playing particularly important roles in SFK activation and neuritogenesis, and Y185 having modifying effects secondary to Y232 and Y220. Our data support a role for all four Dab1 tyrosine phosphorylation sites in mediating the spectrum of activities associated with Reelin-Dab1 signaling in neurons.

Role of heat-shock factor 2 in cerebral cortex formation and as a regulatorof p35 expression

Heat-shock factors (HSFs) are associated with multiple developmental processes, but their mechanisms of action in these processes remain largely enigmatic. Hsf2-null mice display gametogenesis defects and brain abnormalities characterized by enlarged ventricles. Here, we show that Hsf2-/- cerebral cortex displays mispositioning of neurons of superficial layers. HSF2 deficiency resulted in a reduced number of radial glia fibers, the architectural guides for migrating neurons, and of Cajal-Retzius cells, which secrete the positioning signal Reelin. Therefore, we focused on the radial migration signaling pathways. The levels of Reelin and Dab1 tyrosine phosphorylation were reduced, suggesting that the Reelin cascade is affected in Hsf2-/- cortices. The expression of p35, an activator of cyclin-dependent kinase 5 (Cdk5), essential for radial migration, was dependent on the amount of HSF2 in gain- and loss-of-function systems. p39, another Cdk5 activator, displayed reduced mRNA levels in Hsf2-/- cortices, which, together with the lowered p35 levels, decreased Cdk5 activity. We demonstrate in vivo binding of HSF2 to the p35 promoter and thereby identify p35 as the first target gene for HSF2 in cortical development. In conclusion, HSF2 affects cellular populations that assist in radial migration and directly regulates the expression of p35, a crucial actor of radial neuronal migration.

Modulation of Reelin signaling by Cyclin-dependent kinase 5

The Reelin signaling and Cyclin-dependent kinase 5 (Cdk5) both regulate neuronal positioning in the developing brain. Using double-transgenic mice, we have previously shown that these two signaling pathways lie in parallel fashion and have a genetic interaction. Disabled-1 (Dab1), an adapter protein, mediates Reelin signaling and becomes tyrosine-phosphorylated on the binding of Reelin to its receptors. Several isoforms of Dab1 are expressed in embryonic mouse brain, and p80 [Dab1(555)] is the major protein translated. In the present study, we investigated whether Cdk5-mediated phosphorylation of Dab1 modulates Reelin signaling. Cdk5 phosphorylates p80 Dab1 at multiple sites in its carboxyl-terminal region, and tyrosine phosphorylation of p80 Dab1 by Fyn tyrosine kinase is attenuated by this Cdk5-mediated phosphorylation in vitro. Tyrosine phosphorylation of p80 Dab1 induced by exogenous Reelin is enhanced in Cdk5-deficient neurons, corroborating the inhibitory effect of Cdk5-mediated Ser/Thr phosphorylation on tyrosine phosphorylation of p80 Dab1. Another isoform, p45 Dab1 [Dab1(271)], however, is phosphorylated by Cdk5 at one serine residue within a unique carboxyl-terminal region, and its serine phosphorylation enhances tyrosine phosphorylation by Fyn and results in progressive degradation of p45 Dab1. These results indicate that Cdk5 modulates Reelin signaling through the Ser/Thr phosphorylation of Dab1 differently in an isoform-specific manner.

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.

Absence of Fyn and Src Causes a Reeler-Like Phenotype

Nonreceptor protein tyrosine kinases of the Src family regulate the survival, proliferation, differentiation, and motility of many cell types, but their roles in brain development are unclear. Biochemical and in vitro experiments implicate Src and Fyn in the Reelin-dependent tyrosine phosphorylation of Dab1, which controls the positioning of radially migrating neurons in many brain regions. However, genetic evidence that either Src or Fyn mediates Reelin-dependent migrations in vivo has been lacking. Here, we report that, although Src is dispensable and although the absence of Fyn causes an intermediate phenotype, the combined absence of Src and Fyn almost abolishes tyrosine phosphorylation of Dab1 and causes defects in the fetal cortex and cerebellum very similar to those of dab1 mutants of the same age. Neurogenesis is not detectably affected, but the layering of neurons in the cortex is inverted, and the formation of the Purkinje plate is impaired. This implies that Src and Fyn are needed for Reelin-dependent events during brain development.