Brian W. Howell

DIRECT BINDING OF REELIN TO VLDL RECEPTOR AND APOE RECEPTOR 2 INDUCES TYROSINE PHOSPHORYLATION OF DISABLED-1 AND MODULATES TAU PHOSPHORYLATION

The large extracellular matrix protein Reelin is produced by Cajal-Retzius neurons in specific regions of the developing brain, where it controls neuronal migration and positioning. Genetic evidence suggests that interpretation of the Reelin signal by migrating neurons involves two neuronal cell surface proteins, the very low density lipoprotein receptor (VLDLR) and the apoE receptor 2 (ApoER2) as well as a cytosolic adaptor protein, Disabled-1 (Dab1). We show that Reelin binds directly and specifically to the ectodomains of VLDLR and ApoER2 in vitro and that blockade of VLDLR and ApoER2 correlates with loss of Reelin-induced tyrosine phosphorylation of Disabled-1 in cultured primary embryonic neurons. Furthermore, mice that lack either Reelin or both VLDLR and ApoER2 exhibit hyperphosphorylation of the microtubule-stabilizing protein tau. Taken together, these findings suggest that Reelin acts via VLDLR and ApoER2 to regulate Disabled-1 tyrosine phosphorylation and microtubule function in neurons.

Neuronal position in the developing brain is regulated by mouse disabled-1.

During mammalian brain development, immature neurons migrate radially from the neuroectoderm to defined locations, giving rise to characteristic cell layers,. Here we show that targeted disruption of the mouse disabled1 ( mdab1 ) gene disturbs neuronal layering in the cerebral cortex, hippocampus and cerebellum. The gene encodes a cytoplasmic protein, mDab1 p80, which is expressed and tyrosine-phosphorylated in the developing nervous system. It is likely to be an adaptor protein, docking to others through its phosphotyrosine residues and protein-interacting domain. The mdab1 mutant phenotype is very similar to that of the reeler mouse. The product of the reeler gene, Reelin, is a secreted protein that has been proposed to act as an extracellular signpost for migrating neurons. Because mDab1 is expressed in wild-type cortical neurons, and Reelin expression is normal in mdab1 mutants, mDab1 may be part of a Reelin-regulated or parallel pathway that controls the final positioning of neurons.

Scrambler and yotari disrupt the disabled gene and produce a reeler- like phenotype in mice

Formation of the mammalian brain requires choreographed migration of neurons to generate highly ordered laminar structures such as those in the cortices of the forebrain and the cerebellum. These processes are severely disrupted by mutations in reelin which cause widespread misplacement of neurons and associated ataxia in reeler mice,. Reelin is a large extracellular protein secreted by pioneer neurons that coordinates cell positioning during neurodevelopment,. Two new autosomal recessive mouse mutations, scrambler and yotari have been described that exhibit a phenotype identical to reeler. Here we report that scrambler and yotari arise from mutations in mdab1 (ref. 12), a mouse gene related to the Drosophila gene disabled ( dab ). Both scrambler and yotari mice express mutated forms of mdab1 messenger RNA and little or no mDab1 protein. mDab1 is a phosphoprotein that appears to function as an intracellular adaptor in protein kinase pathways. Expression analysis indicates that mdab1 is expressed in neuronal populations exposed to Reelin. The similar phenotypes of reeler, scrambler, yotari and mdab1 null mice indicate that Reelin and mDab1 function as signalling molecules that regulate cell positioning in the developing brain.

The when and how of Src regulation

Jonathan A. Cooper and Brian Howell Fred Hutchinson Cancer Research Center Seattle, Washington 98104 With the discovery of yrk (Sudol et al., 1993). there are now nine members of the src gene family. Alternative translational initiation codons and tissue-specific splicing result in more than 14 different src-related gene products being selectively expressed in various cell types (reviewed by Cooper, 1990; Bolen et al., 1992). These proteins are all protein-tyrosine kinases. The family resemblances extend over all but the first 60-80 residues of the total length of around 500-530 residues. The conserved regions can be divided into five sequence blocks. From the N-terminus, these are the extreme N-terminal myristoylation signal, the Src homology (SH) 3 and SH2 regions, the kinase domain, and the C-terminal noncatalytic tail. The structural similarities allow for common methods of regulation. In this essay we focus on the structures and mechanisms that confer negative regulation and discuss how inhibition is relaxed or overridden to allow activation. Repressed and Activated Forms of Src An indication of a possible mechanism of reversible regu- lation of Src in the cell came from the observation that Src is activated by phosphatases present in cell lysates (Courtneidge, 1985). An inhibitory tyrosine phosphoryla- tion site was identified in the C-terminal tail, Tyr-527 in Src (Cooper et al., 1986) which is conserved in other family members. In most naturally occurring, activated mutants of Src, the C-terminal tyrosine is either missing or under- phosphorylated compared with wild type, and a tyrosine in the catalytic domain (residue 416) becomes more highly phosphorylated (reviewed by Jove and Hanafusa, 1987). In vitro experiments show that changes in phosphorylation induce changes in activity: C-terminal phosphotylation is inhibitory, and kinase domain phosphorylation is stimula- tory (Kmiecik et al., 1988). The different phosphorylations can be viewed as stabilizing either an activated or a re- pressed conformation of Src, altering the relative free en- ergies of these states (see Figure 1, bottom). Each confor- mation is stable, but the molecule can flip between them via a small energy barrier. In the cell, there appears to be rapid turnover of phosphate at both residues, so the molecule would be free to “breathe” between the two pro- posed conformations. For Src-related kinases, the energy diagrams would have the same general form, but the shapes would be different to account for different relative effects of the stimulatory and inhibitory phosphorylations. The mutations that activate Src have been mapped to many sites in the kinase domain, SH2 and SH3 domains, and the C-terminal tail (reviewed by Parsons and Weber, 1989). This suggests that the normal, repressed state is maintained by cooperative intramolecular interactions in- volving the entire conserved part of the protein. Activating mutations in the divergent N-terminal region have not been described, so this region is probably not integrated into

The Disabled 1 Phosphotyrosine-Binding Domain Binds to the Internalization Signals of Transmembrane Glycoproteins and to Phospholipids

ABSTRACT Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 μM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.

Mouse disabled (mDab1): a Src binding protein implicated in neuronal development

Here, we identify a mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, and show it is an adaptor molecule functioning in neural development. We find that mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine‐phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine‐containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks.

Thyroid hormone regulates reelin and dab1 expression during brain development

The reelin and dab1 genes are necessary for appropriate neuronal migration and lamination during brain development. Since these processes are controlled by thyroid hormone, we studied the effect of thyroid hormone deprivation and administration on the expression of reelin anddab1. As shown by Northern analysis, in situ hybridization, and immunohistochemistry studies, hypothyroid rats expressed decreased levels of reelinRNA and protein during the perinatal period [embryonic day 18 (E18) and postnatal day 0 (P0)]. The effect was evident in Cajal-Retzius cells of cortex layer I, as well as in layers V/VI, hippocampus, and granular neurons of the cerebellum. At later ages, however, Reelin was more abundant in the cortex, hippocampus, cerebellum, and olfactory bulb of hypothyroid rats (P5), and no differences were detected at P15. Conversely, Dab1 levels were higher at P0, and lower at P5 in hypothyroid animals. In line with these results, reelin RNA and protein levels were higher in cultured hippocampal slices from P0 control rats compared to those from hypothyroid animals. Significantly, thyroid-dependent regulation of reelin anddab1 was confirmed in vivo and in vitro by hormone treatment of hypothyroid rats and organotypic cultures, respectively. In both cases, thyroid hormone led to an increase in reelin expression. Our data suggest that the effects of thyroid hormone on neuronal migration may be in part mediated through the control of reelin anddab1 expression during brain ontogenesis.

Csk suppression of Src involves movement of Csk to sites of Src activity.

Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.

Lipoprotein Receptors: Signaling Functions in the Brain?

brain has a characteristic architecture of packed layers of cells with specific functional identities. In VLDLR Ϫ apoER2 Ϫ , reln Ϫ , and dab1 Ϫ brains, these layers are disorganized. For example, the Purkinje cells of the cerebellum normally migrate outward from an origin near the fourth While some nutrients enter cells through transporters, ventricle, to form a thin layer beneath cerebellar granule others, including iron and cholesterol, are actively im-cell precursors. In the mutants, the Purkinje cells initiate ported into cells by specialized receptors. These import migration but remain clustered deeper in the cerebellar receptors continuously recycle between the cell surface primordium (Lambert de Rouvroit and Goffinet, 1998; and intracellular vesicles. Cargo is carried from the cell Trommsdorff et al., 1999). Since the proliferation of gran-surface via clathrin-coated pits to endosomes, acidified ule cells requires mitogens, such as Sonic hedgehog, compartments where the cargo is released. The recep-made by the Purkinje cells, the number of granule cells tors then return to the cell surface for another cycle of is markedly reduced in the mutants, and the mutant import. One import receptor, the low density lipoprotein cerebella are about five times smaller than usual. receptor (LDLR), has been intensively studied because The development of the cortex is orchestrated differ-its malfunction is a cause of atherosclerosis. The LDLR ently (Pearlman et al., 1998). Early neurons form two is essential to transport LDL (complexes of cholesterol, layers, an inner subplate and an outer layer of Cajal-triglycerides, and specific apolipoproteins) out of the Retzius (CR) neurons. Later neurons, which will form the plasma, as first shown by the hallmark analysis of familial cortical plate of the embryonic cortex, move through hypercholesterolemia by Brown and Goldstein. In sub-the subplate layer and come to rest below the CR cells. sequent years, other import receptors related to the As each subsequent cortical plate neuron migrates, it LDLR have been discovered, and the LDLR superfamily bypasses its predecessors, coming to rest immediately now includes at least five mammalian and several inver-below the CR neurons. In the mutants, the migrating tebrate proteins. It has become clear that most of the cells neither penetrate between subplate neurons nor new members of the family do not have a primary func-overtake their brethren (Pearlman et al., 1998; Rice et tion in LDL import, and instead bind and import multiple al., 1998). The CR, subplate, and early cortical plate ligands (see …

Cerebellar abnormalities in the disabled (mdab1–1) mouse

A mouse homolog of the Drosophila Disabled (dab) gene, disabled‐1 (mdab1), encodes an adaptor molecule that functions in neural development. Targeted disruption of the mdab1 gene (mdab1–1 mice) leads to anomalies in the development of the cerebrum, hippocampus, and cerebellum. Here we describe a number of histologic abnormalities in the cerebellum of the mdab1–1 mouse. There is a complete absence of foliation, and most Purkinje cells are clumped in central clusters. However, lamination appears to develop normally in areas where the Purkinje cells and external granular layer are closely apposed. The granular layer forms a thin rind over most of the cerebellar surface, but is subdivided by both transverse and parasagittal boundaries. The Purkinje cells, identified by anti‐zebrin II in the adult or anti‐calbindin in the new born mdab1–1 mutant cerebellum, form a parasagittal banding pattern, similar to but distorted compared with the wild‐type design. The data suggest that the development of the mdab1–1 cerebellum parallels the development of reeler. The reeler gene encodes an extracellular protein (Reelin) that is secreted by the external granular layer. Because Reelin expression is retained in the mdab1–1 mutant mouse, mDab1 p80 may act in a parallel pathway or downstream of Reelin, leading to the transformation of embryonic Purkinje cell clusters into the adult parasagittal bands. J. Comp. Neurol. 402:238–251, 1998.