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Cullin 5 Regulates Dab1 Protein Levels and Neuron Positioning During Cortical Development

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Cullin 5 Regulates Dab1 Protein Levels and Neuron Positioning During Cortical Development Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Cullin 5 regulates Dab1 protein levels and neuron positioning during cortical development Libing Feng,1,3 Nathaniel S. Allen,1,2,3 Sergi Simo,1 and Jonathan A. Cooper1,2,4 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA; 2Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center and University of Washington, Seattle, Washington 98109, USA Many laminated regions of the mammalian brain develop by the migration of neuronal precursor cells, whose final positions are coordinated by signals from the secreted molecule Reelin. Early events in Reelin signaling have been identified, but the mechanism of signal down-regulation has been unclear. A possible source of negative feedback is the Reelin-induced degradation of the critical intracellular signaling component, Disabled-1 (Dab1). Here we show that degradation of Dab1 depends on Dab1 phosphorylation at specific tyrosine residues and on the E3 ubiquitin ligase component Cullin 5 (Cul5). Cul5 forms complexes with SOCS (suppressors of cytokine signaling) proteins, which bind to phosphorylated Dab1 and target it for degradation in tissue culture cells. Ablation of Cul5 in migrating neurons causes an accumulation of active Dab1 protein and a unique cortical layering defect, characterized by excess migration and buildup of neurons at the top of the cortical plate. The results implicate Cul5 and SOCS proteins in down-regulation of Dab1 in vivo and show that Cul5 plays an essential role in regulating neuron migrations during cortical development, possibly by opposing a promigratory effect of Dab1. [Keywords: Cullin; Dab1; Reelin; cortical development; neuronal migration; ubiquitin] Supplemental material is available at http://www.genesdev.org. Received August 13, 2007; revised version accepted September 14, 2007. Development of complex organisms requires precise pro- nisms to direct the migration of each new layer of neu- duction, positioning, and differentiation of many cell rons past its predecessors (Rakic 1995; Kriegstein et al. types. In some cases, cells migrate long distances and 2006). As the cortex grows, neurons fated for the upper along intricate routes to reach the places where they dif- cortical plate layers must navigate through a thicket of ferentiate. This is the case during development of the other cells. mammalian neocortex (Hatten 2002; Marin and Ruben- The neuron migrations that organize this inside-out stein 2003; Nadarajah et al. 2003; Kriegstein and Noctor layering of the neocortex are regulated by the Reelin– 2004). Neuronal progenitor cells, located in the ventricu- Dab1 pathway (for review, see Rice and Curran 2001; lar zone adjacent to the lateral ventricle, give rise to pre- Gupta et al. 2002; Jossin 2004; Herz and Chen 2006). cursors that migrate radially outward through a cell-poor Reelin is a secreted protein that acts on other cells by region called the intermediate zone to establish the ini- inducing clustering of specific receptors, increasing the tial layers of the cortical plate. As embryonic develop- activity of two Src family tyrosine kinases (SFKs), Fyn ment progresses, neuronal precursors will give rise to and Src, and increasing tyrosine phosphorylation of the distinct types of projection neurons—distinguishable by membrane-associated cytoplasmic protein Disabled-1 their gene expression, morphology, axonal and dendritic (Dab1). All these components have been shown by ge- architecture, and synaptic properties—that arrange them- netics and biochemistry to be involved in Reelin signal- selves into six discrete layers that compose the mature ing in the neocortex, and failure of Reelin to induce Dab1 cortical plate. These layers are normally arranged in an tyrosine phosphorylation during development results in inside-out manner according to neuronal birth order. an inversion of cortical plate layering. This arrangement is thought to have allowed for expan- While much is known about the initial events in sion of the neocortex during mammalian evolution, a Reelin signaling, less is known about how this signal is process that would also require the evolution of mecha- modulated. Negative feedback buffers signaling against noise and allows for adaptation, which is important for cells or axons to follow a concentration gradient during 3These authors contributed equally to this work. chemotaxis or to respond appropriately to temporal 4Corresponding author. E-MAIL [email protected]; FAX (206) 667-6522. changes in stimulus strength (Ferrell 1996; Yi et al. 2000; Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1604207. Heinrich et al. 2002; Ming et al. 2002; Weiner 2002). GENES & DEVELOPMENT 21:2717–2730 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org 2717 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Feng et al. While many signaling pathways are down-regulated by 2005). We were interested in which phosphotyrosines in inactivation of a kinase, SFKs that have been activated Dab1 might be involved in turnover. Dab1 can be phos- by Reelin remain active due to positive feedback from phorylated at up to four sites—a (Tyr185), b (Tyr198), phospho-Dab1 (Arnaud et al. 2003b; Ballif et al. 2003; c (Tyr220), and d (Tyr232)—by forced expression of an Bock and Herz 2003). However, Reelin-induced tyrosine SFK (Fig. 1A; Howell et al. 2000; Keshvara et al. 2001). phosphorylation of Dab1 leads to polyubiquitination and Phosphorylation of the b, c, and d sites has been de- proteasome-dependent degradation of Dab1 (Arnaud et tected in neurons (Keshvara et al. 2001; Ballif et al. al. 2003a; Bock et al. 2004). Due to this degradation, 2004). The a and b sites lie in similar YQXI sequences, Dab1 protein levels are lower in wild-type embryo brains and the c and d sites lie in YXVP. To allow for pos- than in Reelin, receptor, or SFK mutants (Rice et al. sible redundancy between sites of similar sequence, we 1998; Trommsdorff et al. 1999; Howell et al. 2000; Kuo compared the Reelin-induced phosphorylation and et al. 2005). After Reelin-induced down-regulation, Dab1 degradation of wild-type Dab1WT, Dab1ab (sites a and b protein levels recover slowly, providing a refractory pe- mutated), Dab1cd (sites c and d mutated), and Dab15F riod during which the cells cannot respond a second time (mutated at all four sites and Tyr200) (Howell et al. (Arnaud et al. 2003a; Bock et al. 2004). Reelin-induced 2000). These proteins were tagged with GFP to distin- degradation is a major mechanism for signal down-regu- guish them from endogenous Dab1, and expressed in pri- lation, because proteasome inhibitors cause a sustained mary neuron cultures using electroporation (Xu et al. tyrosine phosphorylation of Dab1 in response to Reelin. 2005). These observations raise the possibility that degradation As shown in Figure 1B, phosphorylation of wild-type of tyrosine-phosphorylated Dab1 is a negative feedback Dab1WT-GFP is stimulated by Reelin, as expected. mechanism that could be important for regulating Ree- Dab1ab-GFP and Dab1cd-GFP proteins exhibit reduced lin responses in vivo. levels of basal and Reelin-stimulated tyrosine phos- Here we investigated the mechanism for Dab1 degra- phorylation, but in both cases, Reelin still stimulates dation. Previous reports had shown that overexpres- some phosphorylation of the remaining sites (Fig. 1B). sion of the E3 ubiquitin ligase Cbl induces Dab1 ubiqui- This means that neither pair of phosphorylation sites is tination in tissue culture cells (Suetsugu et al. 2004). essential for phosphorylation of the remaining sites, at However, degradation of the ubiquitinated Dab1 was not least when the proteins are overexpressed. reported. In addition, Dab1 was found to bind and inhibit We then tested for Reelin-induced degradation, using another E3 ligase, Siah-1A (Park et al. 2003). In contrast, MAP2-GFP as an internal control for electroporation we found that degradation of Dab1 in tissue culture cells efficiency. Cultures were exposed to Reelin for 3–5d is not induced by Cbl, but is specifically stimulated by to induce Dab1 down-regulation. We found that overexpression of suppressor of cytokine signaling-1 Dab1WT-GFP is down-regulated in Reelin-stimulated (SOCS1) or SOCS3 proteins. We identified the tyrosine cultures relative to control cultures (Fig. 1C). By com- residues in Dab1 that mediate Reelin-induced Dab1 paring the level of Dab1WT-GFP with MAP2-GFP in re- down-regulation in neurons and found that the same peated experiments, we determined that the Reelin- sites mediate SOCS-stimulated degradation in tissue induced decrease was ∼30% (Fig. 1C, histogram). This culture cells. SOCS proteins are adaptors for an E3 ligase compares with ∼80% decrease in endogenous Dab1 p80 complex containing the key component Cullin-5 (Cul5) in the same cultures (Fig. 1C, middle panel). The in- (for review, see Ilangumaran et al. 2004; Petroski and efficient degradation of Dab1WT-GFP relative to endog- Deshaies 2005). Knockdown of Cul5 in cultured neurons enous Dab1 p80 may be due to its overexpression and protects active Dab1 from Reelin-induced degradation. lower stoichiometry of phosphorylation (Xu et al. 2005). Furthermore, Cul5 knockdown in developing mouse However, the degradation of Dab1WT-GFP required phos- brain causes accumulation of phosphorylated, active phorylation, since levels of Dab15F-GFP were equal in Dab1 in migrating neurons, confirming the importance Reelin-treated and control cultures (Fig. 1C). More- of Cul5 for Dab1 degradation in vivo. Cul5 knockdown over, Dab1ab-GFP was not degraded in response to also causes a novel upward shift in positions of cortical Reelin, while Dab1cd-GFP was degraded to the same ex- neurons, suggesting that ubiquitin-mediated proteolysis tent at Dab1WT-GFP. Testing the a and b phosphoryla- plays an important role in cortical lamination. This pro- tion sites individually showed that the b site is required vides the first evidence that protein degradation is in- for Reelin-induced degradation (Fig.
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