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provided by Elsevier - Publisher Connector Current Biology, Vol. 14, 606–610, April 6, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.03.038 Activation of a Dab1/CrkL/C3G/Rap1 Pathway in -Stimulated Neurons

Bryan A. Ballif,1,3 Lionel Arnaud,1,4 containing nonphosphorylated and tyrosine-phosphor- William T. Arthur,1 Deborah Guris,2 ylated Dab1 (Figure 1A). The tyrosine-phosphorylated Akira Imamoto,2 and Jonathan A. Cooper1,* Dab1 was a truncated form, which resembles the func- 1Fred Hutchinson Cancer Research Center tional p45 splice variant of Dab1 [15], and nonphosphor- 1100 Fairview Avenue North ylated Dab1 was the corresponding mutant with four Seattle Washington 98109 potential tyrosine phosphorylation sites mutated to phe- 2 The Ben May Institute for Cancer Research and nylalanine. N- and C-terminal his6 and GST tags ensured Center for Molecular Oncology that fusion proteins were intact. After extensive washing, University of Chicago proteins on the various columns were eluted with mag- Chicago, Illinois 60637 nesium chloride. Subsequent elution of the columns with SDS showed that the fusion proteins remained intact and highly phosphorylated on tyrosine (Figure 1B). Summary Brain proteins bound to each column were analyzed by SDS polyacrylamide gel electrophoresis (Figure 1C). During brain development, many neurons migrate long Several proteins were enriched on the tyrosine-phos- distances before settling and differentiating. These mi- phorylated Dab1 column (c). These gel bands were ex- grations are coordinated to ensure normal develop- cised and proteins in them identified by using mass ment. The secreted protein Reelin controls the loca- spectrometry (see Supplemental Data; Table S1). We tions of many types of neurons, and its absence causes confirmed that PLC-␥1, Shp2, Src, CrkL, CrkII, and CrkI the classic “” phenotype [1, 2]. Reelin action bind specifically to tyrosine-phosphorylated, but not requires tyrosine phosphorylation of the intracellular nonphosphorylated, Dab1 in vitro by Western blotting protein Dab1 by Src-family kinases [3–5]. However, the column fractions (Figure S1). The relative abundance little is known about signaling pathways downstream of these proteins in the column eluate (Figure 1C) likely of Dab1. Here, we identify several proteins in embry- reflects both their relative abundance in E16.5 mouse onic brain extract that bind to tyrosine-phosphory- brain extract and their relative affinities for tyrosine- lated, but not non-phosphorylated, Dab1. Of these, the phosphorylated Dab1. Crk-family proteins (CrkL, CrkI, and CrkII [6]), bind We tested whether proteins that bind to phosphory- significant quantities of Dab1 when embryonic cortical lated Dab1 in vitro also bind in vivo. Neurons were cul- neurons are exposed to Reelin. CrkL binding to Dab1 tured from E16.5 cortex and exposed to Reelin for 15 involves two tyrosine phosphorylation sites, Y220 and min to stimulate the tyrosine phosphorylation of Dab1 232, that are critical for proper positioning of migrating [4, 16]. Dab1 was readily detected in CrkL and Crk immu- cortical plate neurons. CrkL also binds C3G, an ex- noprecipitates made from Reelin-stimulated neurons change factor (GEF) for the small GTPase Rap1 that is (Figure 2B, lanes 2 and 4). Less Dab1 was complexed activated in other systems by tyrosine phosphorylation with CrkL and Crk prior to Reelin stimulation (Figure 2B, [7–10]. We report that Reelin stimulates tyrosine phos- lanes 1 and 3). Dab1 that was precipitated with CrkL phorylation of C3G and activates Rap1. C3G and Rap1 antibodies from Reelin-treated and control neurons con- regulate adhesion of fibroblasts and other cell types tained phosphotyrosine (Figure S2). After correcting for [11–14]. Regulation of Crk/CrkL, C3G, and Rap1 by antibody efficiency, we estimate that CrkL was associ- Reelin may be involved in coordinating neuron migra- ated with 25% of the tyrosine-phosphorylated Dab1 tions during brain development. present in the lysate. Tyrosine phosphorylation was im- portant for binding because PP2, which inhibits Dab1 Results and Discussion tyrosine phosphorylation [4, 5], ablated Dab1-CrkL com- plexes (Figure S2). In addition, nonphosphorylated Dab1 To identify proteins that might be important for Dab1 protein in neurons made from mouse embryos homozy- signaling in vivo, we screened for embryonic brain pro- gous for a Dab1 allele (Dab15F) did not associate with teins that bind to tyrosine-phosphorylated Dab1 in vitro. Crk or CrkL (Figure 2C, lane 4). CrkL is also associated We reasoned that this approach would be most likely with endogenous Dab1 in vivo, in embryonic brain ex- to identify proteins that both have a high affinity for tracts (Figure S3). Complex formation in vivo also re- tyrosine-phosphorylated Dab1 and are abundant in em- quires Dab1 tyrosine phosphorylation, since CrkL was bryonic brain. Proteins were solubilized from mouse not bound to the Dab15F protein (Figure 2D). Therefore, embryo brains during cortical plate development (em- Dab1 tyrosine phosphorylation is required for binding bryonic day [E]16.5) and purified through several preab- sorption steps before passing over parallel columns to Crk and CrkL in vivo and in Reelin-stimulated neurons. CrkII and CrkL are products of related genes (Crk and CrkL/Crkol) and contain an SH2 domain and two SH3 *Correspondence: [email protected] domains (Figure 2A) [17]. CrkI is an alternatively spliced 3 Present address: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. form of CrkII that lacks the more C-terminal SH3 domain. 4 Present address: Sereno Pharmaceutical Research Institute, Plan- Crk and CrkL are also regulated by tyrosine phosphory- les-Oates, Switzerland. lation [17–21]. However, CrkL tyrosine phosphorylation Reelin Activates C3G and Rap1 via Dab1 607

did not change detectably after Reelin stimulation (Fig- ure S2). SH2 domains bind to phosphotyrosine in specific se- quence contexts [22]. We confirmed that Dab1 protein from embryonic brain extracts bound in vitro to a GST fusion protein containing the SH2 domain, but not the SH3 domain, of CrkL (Figure S4). To test which Dab1 tyrosine phosphorylation sites are involved, Dab1 was tagged with GFP and expressed in tissue culture to- gether with the tyrosine kinase Fyn. GFP-Dab1 bound to GST-CrkL-SH2 only when tyrosine phosphorylated (Figure 3A). GFP-Dab1 proteins containing at single phosphorylation sites were tested in the same manner (Figure 3B). While of Y198, Y185, or both had little effect on binding (Figure 3B, lanes 2, 3, and 6), mutation of either Y220 or Y232 inhibited binding, even though both these single mutants were still highly phosphorylated at other tyrosine residues (lanes 4 and 5). All combinations of mutations that included either Y220F (lanes 7, 8, 10, 11, and 12) or Y232F (lane 7, 9, 11, and 12) were greatly inhibited for binding. The importance of Y232 was surprising since Y232 is report- edly not phosphorylated in response to Reelin [23]. How- ever, we reinvestigated the phosphorylation status of Dab1 in Reelin-stimulated neurons and found evidence Figure 1. Purification of Murine Embryonic Brain Proteins Inter- that both Y220 and Y232 are phosphorylated (Figure acting Specifically with Phosphotyrosyl-Dab1 S5). These results imply that phosphorylation of both (A) Purification scheme (see Supplemental Experimental Proce- Y220 and Y232 is required for Dab1 binding to the CrkL dures). 150 mg of E16.5 brain extract was cleared over a column of SH2 domain. Both sites fit the consensus for binding glutathione Sepharose and then passed sequentially over his6-GST 1–252,4F Crk-family SH2 domains [24], but the requirement for (column a) and his6-Dab1 -GST (column b). The flow-through 1–252,WT two tyrosine residues was unexpected. was then divided equally and applied in parallel to his6-Dab1 - 1–252,4F GST (column c) and his6-Dab1 -GST (column d). After washing, The Rap1 GEF C3G (Figure 4A) is one of several pro- proteins bound to columns a–d were eluted with 1 M MgCl2 and teins that binds to Crk family SH3 domains [6]. C3G is precipitated with trichloroacetic acid. present in embryonic brain and associated with the CrkL (B) After elution, the columns were washed and then stripped with SH3 domain (Figures 2D and S4). Various conditions that hot SDS-PAGE sample buffer. Proportional fractions of the stripped lead to Rap1 activation (including Crk overexpression, columns were subjected to SDS-PAGE and Western blotting. integrin ligation, and growth hormone stimulation) in- (C) MgCl2 eluates from columns a–d were subjected to SDS-PAGE, and the gel was stained with Coomassie blue. Bands denoted by duce tyrosine phosphorylation of C3G [7, 25, 26]. Tyro- asterisks were cut from the gel and identified by trypsin digest and sine phosphorylation site Y504 is required for activation LC-MS/MS (Table S1). of Rap1 by C3G [7] (Figure 4A). Therefore, we tested whether C3G is tyrosine phosphorylated in response to Reelin stimulation. Reelin stimulated the tyrosine phos-

Figure 2. Dab1-Crk Family Complexes in Pri- mary Neuronal Cultures and Developing Brain (A) Domain structure and percent amino acid identity between SH2 and SH3 domains of Crk family members. Dashed lines between CrkI and CrkII indicate alternative splicing. (B) E16.5 cultures were ex- posed to either mock (M)- or Reelin (R)-con- taining supernatant for 15 min. Cells were then lysed and analyzed by immunoprecipita- tion and Western blotting. The band below Dab1 is nonspecific. (C) Primary cortical cultures from Dab1ϩ/ϩ or Dab15F/5F littermates were treated and ana- lyzed similarly. (D) E16.5 brains of Dab1ϩ/ϩ and Dab15F/5F lit- termates were analyzed by immunoprecipita- tion and Western blotting. Current Biology 608

Figure 3. The SH2 Domain of CrkL Binds to Phosphorylated Tyrosine Residues 232 and 220 of Dab1 (A) HEK 293T cells were cotransfected with constructs encoding full-length Dab1 or Dab15F fused to EGFP and either activated Fyn (FynY531F) or kinase-deficient Fyn (FynK299M) as indicated. Extracts were incubated with the SH2 domain of CrkL fused to GST. Bound proteins (pulldowns) and extracts were ana- lyzed by Western blot. (B) HEK 293T cells were cotransfected with constructs encoding activated FynY531F and ei- ther wild-type or tyrosine to phenylalanine mutants of EGFP-Dab1. The letters (a–d) rep- resent the tyrosine residues mutated to phe- nylalanine in each mutant. Cell extracts and GST-CrkL-SH2 pulldowns were analyzed by Western blot.

phorylation of C3G, but not another Crk binding protein We assayed Rap1.GTP levels by measuring Rap1 (p130Cas) (Figure 4B). Tyrosine phosphorylation of C3G binding to a Rap1 binding domain, RalGDS-RBD, in vitro was not detected in neurons expressing the nonphos- [27]. This assay shows the expected increase in phorylated Dab15F (Figure 4C). Therefore, C3G is tyrosine Rap1.GTP when C3G was overexpressed in HeLa cells phosphorylated in response to Reelin, provided that (Figure 4D, lanes 1–2). We measured the effects of Reelin Dab1 is also phosphorylated. on Rap1.GTP levels in three independent preparations

Figure 4. Reelin Induces C3G Tyrosine Phos- phorylation and Rap1 Activation (A) C3G structure. (B) Primary cortical cultures were treated with mock (M)- or Reelin (R)-containing superna- tants for 15 min. Cells were lysed and C3G immunoprecipitated. Immune complexes and neuronal extracts were Western blotted with the indicated antibodies. (C) Primary cortical cultures from Dab1ϩ/ϩ or Dab15F/5F littermates were treated with mock (M)- or Reelin (R)-containing supernatants for 15 min. Cells were lysed and C3G immuno- precipitated. (D) Rap1 GTP loading. Lanes 1 and 2, HeLa cells were transiently transfected with (1) vec- tor or (2) C3G. Lysates were analyzed directly or after pulldown with GST-RalGDS-RBD to purify Rap1.GTP. Samples were immunoblot- ted with anti-Rap1 antibodies. The GTP con- tent of Rap1 was increased by C3G, as ex- pected. Lanes 3 and 4, E16.5 neurons were treated with Mock (M) or Reelin (R)-containing supernatants for 15 min prior to lysis, pull- down with GST-RalGDS-RBD, and immu- noblotting with anti-Rap1 antibody. (E) Histogram of Rap.GTP levels from three experiments, each performed in duplicate. (F) Analysis of Rac1 GTP content using GST- Pak1-CRIB for pulldowns and anti-Rac1 anti- body for immunoblotting. Lanes 1 and 2, transient expression of VAV2 induced an increase in Rac1.GTP in HeLa cells. Lanes 3 and 4, no change in Rac1.GTP was detected in Reelin-stimulated neurons. Reelin Activates C3G and Rap1 via Dab1 609

of neurons. We detected a significant increase in personal communication). Thus the requirements for Rap1.GTP (p Ͻ 0.005, 46% Ϯ 39%, n ϭ 6) after Reelin signaling in vivo and CrkL binding in vitro are congruent. stimulation (Figure 4D, lanes 3 and 4, and Figure 4E). Their results suggest that both Y220 and Y232 are phos- Crk family proteins also bind to the Rac GEF, DOCK180 phorylated in vivo and that a protein whose binding [6]. We assayed whether Rac1 GTP loading was altered requires both sites, like CrkL or Crk, is critical for proper in Reelin-stimulated neurons by binding to the Rac1 neuron placement in vivo. effector protein, Pak1 [28]. While overexpression of a It is possible that Dab1 becomes multiply phosphory- Rac1 GEF in HeLa cells increased Rac1.GTP, there was lated at all four potential phosphorylation sites, perhaps no change in Rac1.GTP levels in Reelin-treated neurons by a processive mechanism [46]. In this case, assembly (Figure 4F). These results show that Reelin induces C3G of multiprotein complexes with several associated SH2- tyrosine phosphorylation and Rap1 activation in cortical containing proteins may be possible. In fact, the surpris- neurons. ing finding that both Y220 and Y232 are needed for We have found that Crk and CrkL associate with a efficient binding of Dab1 to the CrkL SH2 domain in vitro substantial fraction of the Dab1 that becomes phosphor- may be best explained by proposing that individual Dab1 ylated on tyrosine following Reelin stimulation. Dab1 molecules are phosphorylated at both sites and that and CrkL are both expressed throughout the developing CrkL and Crk bind as a dimers, with two SH2 domains cortical plate. Dab1-CrkL complexes could be detected simultaneously contacting a single Dab1. The affinity of in embryonic brain and cultured neurons, depending on such a bivalent interaction would be much stronger than Dab1 tyrosine phosphorylation. Crk proteins are also the binding of a single SH2 domain. While Crk-family bound to C3G, and Reelin stimulates the tyrosine phos- proteins have not been reported to dimerize, C3G con- phorylation of C3G depending on Dab1. Besides Dab1 tains three binding sites for the Crk SH3 domain, and a and SFKs, C3G is the first tyrosine phosphorylation complex containing one C3G, one Dab1, and two Crk- event detected in Reelin-stimulated neurons. Consistent family proteins may form in response to Reelin. with Reelin-induced tyrosine phosphorylation of C3G, Rap1 is activated in Reelin-stimulated neurons. Conclusions Rap1 is a member of the Ras subfamily of GTPases, We provide evidence for a CrkL/C3G/Rap1 pathway op- and like Ras, it is active when bound to GTP and inactive erating downstream of Dab1 in Reelin-stimulated neu- when bound to GDP. In some cell types it competes rons. Since C3G and Rap1 regulate integrin-dependent with the conventional Ras proteins (H-Ras, K-Ras, and adhesion and cell motility, this pathway may be impor- N-Ras) for binding to cRaf1, hence inhibiting MAP kinase tant for proper neuron placement during development. activation [29]. However, in PC12 cells, active Rap1 as- sociates with B-Raf and stimulates MAP kinase [30]. We Supplemental Data have not detected either an increase or a decrease in Supplemental Data including Experimental Procedures, five figures, and one table are available at http://www.current-biology.com/cgi/ MAP kinase activity in Reelin-stimulated neurons [31]. content/full/14/7/606/DC1/. In addition, Rap1 regulates cytoskeletal events. In fibro- blasts, lymphocytes, and myeloid cells, Rap1 stimulates Acknowledgments integrin-mediated adhesion and regulates migration [12, 14, 32–35]. Reelin regulates adhesive events between We are very grateful to Tara Herrick and Priscilla Kronstadt O’Brien neurons, as shown in various ex vivo assays [36–39]. for technical assistance; Li-Huei Tsai for interesting discussions and However, it remains to be investigated whether Rap1 is free exchange of unpublished information; Lawrence Quilliam for plasmids and discussions regarding Rap1 activation; Michelle important for Dab1-dependent positioning of neurons Booden, Tohru Tezaka, and Brian Howell for reagents; and Phil in the developing cortical plate in vivo. Gafken and Angela Norbeck of the FHCRC Proteomics Resource. The functions of Crk and CrkL in brain development Supported by National Cancer Institute Training Grant T32 CA 09657 are unknown. The Crk gene is in a region implicated in to B.A.B., a Ruth L. Kirschstein NRSA F32 NS046492 to W.T.A., and a human neuron migration disorder, together with 14- U.S. Public Health Service award CA41072 from the National Cancer 3-3⑀ and Lis1 [40, 41]. The importance of Crk has not Institute (J.A.C.). been investigated but is likely redundant with Crkl. The Received: February 16, 2004 effects of Crkl mutation in the mouse depend on genetic Accepted: February 17, 2004 background. In the most severe case, mice die of other Published: April 6, 2004 causes before brain development is complete, preclud- ing detection of migration defects [42]. One indication References that CrkL or Crk may be important in vivo is our finding that binding to Dab1 requires both Y220 and Y232. This 1. Rice, D.S., and Curran, T. (2001). Role of the reelin signaling pathway in development. Annu. Rev. is unusual. Other Dab1 binding proteins identified have Neurosci. 24, 1005–1039. different phosphorylation requirements. Lis1 binds to 2. 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