Elsevier

Molecular Brain Research

Volume 117, Issue 2, 7 October 2003, Pages 152-159
Molecular Brain Research

Research report
Tyrosine phosphorylation of Disabled-1 is essential for Reelin-stimulated activation of Akt and Src family kinases

https://doi.org/10.1016/S0169-328X(03)00295-XGet rights and content

Abstract

Reelin is a large secreted signaling protein that is essential for proper positioning of migratory neurons during mammalian brain development. The Reelin signal is transduced into the cell by the lipoprotein receptors VLDLR and ApoER2, leading to tyrosine phosphorylation of the associated intracellular adaptor protein Disabled-1 (Dab1). Tyrosine phosphorylation of Dab1 is essential for responding to Reelin, as knock-in mice expressing a form of Dab1 that cannot be phosphorylated on tyrosine are indistinguishable from mice lacking Reelin, Reelin-receptors or Dab1. Molecular events dependent on Dab1 tyrosine phosphorylation are unknown. However, Reelin has recently been shown to activate the phosphoinositide-3-kinase (PI 3-K)-dependent kinase, Akt, as well as Src family kinases in wild type but not Dab1−/− primary embryonic neuronal cultures. Using pharmacological inhibitors and mice harboring mutant alleles of Dab1, we show here that tyrosine phosphorylation, but not the carboxyl-terminal region, of Dab1 is required for Reelin-induced activation of Akt and Src family kinases. Additionally, although Fyn is an important regulator of Dab1, Fyn deficiency does not prevent acute Reelin-induced Akt activation. Finally, whereas a number of growth factors propagate signals simultaneously through PI 3-K and mitogen-activated protein kinase (MAPK) cascades, we find Reelin does not engage the canonical MAPK cascade. These results define the first molecular events strictly dependent on Reelin-induced Dab1 tyrosine phosphorylation, and suggest that propagation of the Reelin signal is mediated by Akt, substrates of Src family kinases and/or unidentified molecules that share with these a common molecular link to phosphorylated Dab1.

Introduction

Proper development of the mammalian brain requires extensive coordination in the delivery and reception of a number of secreted and cell-tethered signaling molecules. Such orchestration shapes cell fate decisions by influencing migration, differentiation, proliferation and survival. A chief regulator governing the final position of migratory neurons in the developing brain is the large secreted protein, Reelin. Mice deficient in Reelin have malformations in highly layered tissues of the brain such as the cerebral cortex, the cerebellum and the hippocampus [19], [36]. Reelin delivers its molecular information by engaging the lipoprotein receptors ApoE receptor-2 (ApoER-2) and very low density lipoprotein receptor (VLDLR) found on the surface of various neuronal cell types in the developing brain [14], [22]. Although these lipoprotein receptors have no intrinsic kinase activity, their binding to Reelin induces tyrosine phosphorylation of an associated intracellular adaptor protein, Dab1 [25]. Strong evidence suggests Dab1 tyrosine phosphorylation is catalyzed by nonreceptor tyrosine kinases of the Src family [1], [6], but other kinases could also be allowed. Reelin induces Dab1 phosphorylation on at least two of five candidate tyrosine residues [26], [29]. These tyrosine residues lie carboxyl-terminal to a ‘phospho-tyrosine binding’ (PTB) domain that, in the case of Dab1, prefers to bind unphosphorylated NPXY motifs found in molecules such as ApoER2 and VLDLR [27].

The importance of Dab1 tyrosine phosphorylation in Reelin signaling is profound, as knock-in mice homozygous for a Dab1 allele encoding tyrosine to phenylalanine substitutions at all five potential phosphorylation sites (Dab15F) are indistinguishable from mice lacking Reelin, Reelin receptors, or Dab1 [15], [24], [26], [38], [42]. Understanding molecular events dependent on Reelin-induced Dab1 tyrosine phosphorylation is therefore of great interest. Recent studies have implicated downstream transducers of Reelin/Dab1 signaling. We [1] and Bock and Herz [6] have shown that Reelin activates Src family kinases in wild type but not Dab1−/− neuronal cultures, and Beffert et al. [3] have shown Dab1 and either ApoER2 or VLDLR is required for Reelin-induced activation of Akt. Given that Dab1 is essential in the activation of these kinases by Reelin, and that phosphorylation of Dab1 is required for proper Dab1 function, we sought to determine if activation of these kinases was strictly dependent on Reelin-stimulated Dab1 tyrosine phosphorylation.

Section snippets

Mice

All mice used in this study were hybrid C57BL6J/129Sv. The generation of mice with Dab1 mutant alleles was previously described [21], [24], [26]. Subcolonies of fyn−/− mice were derived from parental src+/−, fyn+/−, yes−/− mice described previously [1]. Protocols for genotyping tail DNA by PCR are available upon request. Animal care, husbandry and handling were performed in accordance with federal, state and institutional animal welfare policies.

Primary embryonic neuronal cultures

Cerebral hemispheres from mice at embryonic day

Results

Until recently, the only detectable biochemical event induced by Reelin treatment of embryonic neuronal cultures was tyrosine phosphorylation of Dab1 [25]. Beffert et al. have recently shown that Reelin also activates the serine/threonine kinase Akt by inducing its phosphorylation at serine 473 [3]. Additionally, we and others have shown that Reelin activates Src family kinases [1], [6]. Constitutively active or oncogenic forms of Src family kinases, as well as a number of extracellular stimuli

Discussion

Genetic dissection of mammalian signaling pathways is challenged by the relative complexity of their genomes, their relatively few offspring per generation and the relatively demanding techniques required for their genetic manipulation and analysis. Nevertheless, elucidation of components of Reelin signaling has met with success in mice. This has been greatly aided by the restricted number of genes encoding the Reelin ligand [15], its two receptors [42] and the intracellular adaptor [24], [38],

Acknowledgements

We thank Priscilla Kronstad-O’Brien for her outstanding management of animal husbandry and genotyping. This work was supported by National Cancer Institute MERIT award R37 CA41072 (J.A.C.) and National Cancer Institute Training Grant T32 CA09657-11 (B.A.B.).

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