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

Abstract

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.

The Early Isoform of Disabled-1 Functions Independently of Reelin-Mediated Tyrosine Phosphorylation in Chick Retina

“…1). In previous reports, we have shown that exogenous GFP-Dab1-L but not GFPDab1-E is robustly tyrosine phosphorylated in transfected retinal cells (41)(42).…”

Dual Functions of Dab1 during Brain Development

“…Our finding that the ab mutant is virtually inactive implies that the a site may take over if the b site is unavailable. Also, all four phosphorylation sites are required for overexpressed Dab1 to induce the differentiation of zebrafish retinal precursor cells (26). However, phosphorylation of the a site in neurons has not yet been confirmed.…”

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

“…Primary retinal cultures were prepared from embryonic day (ED) 5 or ED10 chick retinas dissociated with trypsin as previously described [8]. The treatments and transfections were carried out 24 h after plating.…”

“…The N-terminal domain is followed by a tyrosine-rich region, which consists of five highly-conserved tyrosine (Y) residues (Y185, Y198, Y200, Y220 and Y232) corresponding to two consensus Src family kinase (SFK) recognition sites (Y185 and Y198/Y200) and two consensus Abl recognition sites (Y220 and Y232) [5]. At least three of the four tyrosine phosphorylation sites (Y198, Y220 and Y232) are phosphorylated by SFKs and/or involved in the activation of SFKs in cultured neurons upon Reelin stimulation [6][7][8]. Tyrosine phosphorylation of Dab1 is essential for Reelin signaling, as mice expressing Dab1 with substitutions at these five tyrosine residues have neuronal cell positioning defects similar to those observed in Reelin-deficient (reeler) and Dab1−/− mice [9].…”