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Originally published In Press as doi:10.1074/jbc.M413356200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 10, 9671-9677, March 11, 2005
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Phosphoinositide Binding by the Disabled-1 PTB Domain Is Necessary for Membrane Localization and Reelin Signal Transduction*

Peggy C. Stolt{ddagger}§, Ying Chen¶, Pingsheng Liu||, Hans H. Bock¶**{ddagger}{ddagger}, Stephen C. Blacklow{ddagger}§§, and Joachim Herz¶

From the {ddagger}Department of Pathology, Harvard Medical School, Brigham and Womens' Hospital, Boston, Massachusetts 02115, the Departments of Molecular Genetics and ||Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and the **Albert-Ludwigs-Universität Freiburg, Innere Medizin II, Zentrum für Neurowissenschaften, Albertstrasse 23, 79104 Freiburg, Germany

Received for publication, November 29, 2004 , and in revised form, December 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P2 through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the developing mammalian brain, neurons that make up the cerebral cortex must form discrete layers that align in an inverted, "inside-out" manner. This is a complex process in which newly born neurons migrate from the ventricular zone to their correct positions in the neocortex. This critical migration event, as well as those in other regions of the developing brain, is regulated in part by the Reelin signaling pathway (1–3). Binding of the large extracellular molecule Reelin to the ectodomains of the lipoprotein receptors apolipoprotein E receptor-2 (ApoER2)1 or the very low density lipoprotein receptor (VLDLR) leads to phosphorylation of the Disabled-1 (Dab1) adaptor molecule, which is bound to the cytoplasmic tail of the receptors (4–6). Downstream signaling continues through a phosphoinositide (PI) 3-kinase-mediated pathway, involving a direct interaction of the PI 3-kinase regulatory subunit p85 with tyrosine-phosphorylated Dab1 (7). Activation of PI 3-kinase leads to phosphorylation of Akt and GSK3{beta}, resulting in hypophosphorylation of the microtubule-associated protein tau and modulation of the cytoskeleton (4, 8).

Murine Dab1 is a 555-amino acid protein whose expression is limited to the brain (9). Dab1 features an amino-terminal phosphotyrosine binding (PTB) domain, followed by a tyrosine-rich region, and an acidic carboxyl terminus (Fig. 1A) (10). The association between Dab1 and the lipoprotein receptors occurs via binding of the Dab1 PTB domain to the NPXY motif in the receptor tails (11). Reelin-induced phosphorylation then takes place within the tyrosine-rich region, with the most important sites believed to be on Tyr198, Tyr220, and possibly Tyr232 (12, 13). This phosphorylation is carried out by Src family kinases, with Fyn tyrosine kinase as the most likely candidate (14, 15). Phosphorylation of Dab1 is critical for transduction of a Reelin signal, as mutation of the 5 tyrosines in this region to phenylalanine results in a phenotype identical to the Dab1 knockout (12). In addition, loss of phosphorylation on Tyr198 and Tyr232 results in specific defects in migration of developing neurons along radial glial fibers (16). Whereas the tyrosine-rich region of Dab1 is the actual site of phosphorylation, it is the NH2-terminal PTB domain that mediates interaction with the lipoprotein tails and facilitates activation. Thus, the PTB domain is essential for proper Dab1 function.



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  		FIG. 1.
The Disabled-1 PTB domain mutants. A, schematic of murine Disabled-1 showing the NH2-terminal PTB domain followed by the tyrosine-rich region, which includes tyrosines 185, 198, 200, 220, and 232. B, structure of the Dab1 PTB domain ternary complex, demonstrating the locations of the NPXY and PI binding sites. The Dab1 PTB domain is shown as a semi-transparent molecular surface representation (green), whereas the ligands are shown in ball-and-stick representation. The NPXY peptide and the surface residues of the PTB domain in contact with it are lavender. PI 4,5-P2 and residues contacting it are salmon. The plasma membrane is highly schematized. C, residues involved in NPXY peptide binding. Labels of residues mutated in this work are underlined. D, residues involved in PI 4,5-P2 binding. Labels of residues mutated in these studies are underlined. Figures were made using Protein Data Bank coordinates 1NU2 [PDB] .

 
The PTB domain is a member of the pleckstrin homology (PH) domain superfamily, which includes PTB, PH, and Ena-Vasp homology (EVH1) domains, and Ran-binding domains (17). These domains are all structurally related but differ in their ligand binding specificities. Recent structural work on the Dab1 PTB domain has shown that this domain is able to bind to the NPXY peptide motif and to a phosphoinositide head group simultaneously through separate binding sites on opposite faces of the domain (Fig. 1B) (18, 19). Thus, Dab1 shares characteristics both with PTB domains that typically recognize NPXY motifs and with PH domains that bind to phosphoinositides (PIs). Subsequent in vitro binding experiments and mutational analysis have revealed that these two ligand-binding sites are non-cooperative and energetically independent (20).

Because the lipoprotein receptors themselves have no associated kinase activity, the interaction between the Dab1 PTB domain and these receptors most likely functions to recruit Dab1 to a complex containing membrane-associated Src family kinases and other components of Reelin signaling in addition to the lipoprotein receptors. As Dab1 is constitutively associated with the membrane in primary neurons (7), PI binding by the Dab1 PTB domain may be responsible for driving membrane localization and thereby facilitating association with the lipoprotein receptor tails. Nevertheless, the functional role of PI binding by the Dab1 PTB domain and its effect on Reelin signal transduction have not yet been examined.

The mutants described in our previous work (20), which have selective deficiencies in either NPXY or PI binding, but not both, constitute powerful reagents for resolving the distinct in vivo role of PI binding from that of binding to the receptor cytoplasmic tails. To examine the functional importance of the two binding sites, we therefore introduced either native or mutant Dab1 proteins deficient in either PI or peptide binding into primary neuronal cultures by lentiviral transduction. The effect of mutations that eliminated either PI or NPXY binding was assessed by: (i) examining the membrane localization of these mutants, and (ii) evaluating the ability of these mutants to become tyrosine-phosphorylated upon Reelin stimulation. These studies show that PI binding by the Dab1 PTB domain is necessary and sufficient for membrane localization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Lentiviral Constructs—The LL3.7 vector (21) was modified by cutting at the XbaI and EcoRI restriction sites to excise the U6 RNA interference promoter and the CMV-EGFP fragment. A fragment from the pIRES-EGFP vector (Invitrogen) encoding the CMV promoter and a multicloning site, followed by an internal ribosome entry site and EGFP was cut out using the AseI and AflIII sites. This fragment was then blunt ligated into the cut LL3.7 vector to make a modified lentiviral vector containing a CMV promoter, an multicloning site for insertion of the Dab1 gene, and an internal ribosome entry site to express EGFP on a separate mRNA. The K45E (20) and S114T/F158V mutations were introduced into the native Dab1 sequence in a pcDNA3.1 vector with a COOH-terminal 5-Myc tag by QuikChange site-directed mutagenesis (Stratagene). The native and mutant Myc-tagged Dab1 sequences were then subcloned into the EcoRI and SmaI sites in the multicloning site of the modified lentiviral vector. The 5F mutant, with mutations Y185F, Y198F, Y200F, Y220F, and Y232F (12) was a kind gift from Brian Howell, and was first subcloned into the pcDNA3.1-Myc vector and then into the modified lentiviral vector in the same manner. Mutations were verified by dideoxy sequencing.

Isothermal Titration Calorimetry—The murine Disabled-1 PTB domain containing the S114T/F158V mutation and encompassing amino acids 20–175 was PCR amplified from the pcDNA3.1-myc plasmid mentioned above. The PCR product was introduced into the pDEST15 Gateway vector (Invitrogen) downstream of glutathione S-transferase, and the protein was produced and purified as described (19). The isothermal titration calorimetry measurements were carried out at 25 °C with the S114T/F158V Dab1 PTB domain protein at a concentration of 20 µM in 20 mM PIPES buffer, pH 7.0, containing 150 mM NaCl and 0.2 mM dithiothreitol. A 14-residue synthetic peptide from the ApoER2 cytoplasmic tail (Invitrogen, acetyl-TKSMNFDNPVYRKT-amide) was purified by reversed-phase high performance liquid chromatography and dissolved in the identical buffer for this titration. The inositol head group of PI 4,5-P2 (Sigma) was also dissolved in the identical buffer. To perform the titrations, a stock solution of either 2 mM ApoER2 peptide or 140 µM PI 4,5-P2 was added in 7.5-µl increments to the solution containing the S114T/F158V mutant Dab1 PTB domain. To extract the dissociation constant, enthalpy of binding, and calculated entropy, data were plotted and analyzed with the software program Origin 5.0.

Transient Transfections—HEK-293 (CRL-1573, ATCC) cells were grown in low-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin. Cells were transfected with the lentiviral constructs described above, or co-transfected with the lentiviral constructs and a pRc-CMV plasmid expressing human Fyn (obtained from Gottfried Baier, Innsbruck) using the FuGENE 6 transfection reagent (Roche Diagnostics). Cell lysates were harvested 24 h after transfection and analyzed by Western blot.

Preparation of Lentiviral Particles—293FT cells were transfected with the pRSV, pRRE, and pVSV-G plasmids of a 3-vector lentiviral system (22, 23), together with the modified lentiviral vector expressing native or mutant Dab1, or an empty vector control. Forty-eight to 72 h after transfection the cell media were collected and centrifuged at 800 x g for 7 min at 4 °C to remove cell debris. The supernatants were dialyzed against serum-free Dulbecco's modified Eagle's medium/F-12 (Invitrogen) using 100-kDa molecular mass cut-off dialysis membranes (Spectrum Labs). The viral supernatants were then supplemented with 2% B27 (Invitrogen) and were frozen in liquid nitrogen and stored at -80 °C. For the experiment in Fig. 5D, the viral media was concentrated 4–5-fold by spinning at 800 x g in Amicon centrifugal filter units with a 100-kDa molecular mass cut-off (Millipore) to achieve a higher titer. To titer the virus, primary neurons were grown on coverslips, transduced, and fixed with 4% paraformaldehyde 2–3 days after transduction. The percentage of cells expressing GFP was counted under a fluorescence microscope, and the titer was calculated to be 2 x 104-1 x 105 infectious units/ml. This titer proved to be sufficient for the assays used in this study at a multiplicity of infection (m.o.i.) of 0.2–0.5.



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  		FIG. 5.
Fractionation of primary neurons expressing native or mutant Disabled-1 constructs. Wild-type rat neurons or neurons from Dab1 knockout embryos were transduced, fractionated, and analyzed by Western blot to determine the fraction of Dab1 associated with the membrane. S, soluble fraction; P, particulate fraction. Vec, empty vector; WT, wild-type Dab1, Dab1 mutants are identified by their amino acid substitutions. Akt was used as a control for soluble protein, whereas LRP was used as a control for the membrane-containing fraction. A, wild-type (+/+), untransduced mouse neurons analyzed for native Dab1 localization using an {alpha}-Dab1 antibody. B, expression of empty vector, wild-type Dab1, K45E, or S114T/F158V mutant constructs in wild-type primary neurons prior to fractionation. Expression of lentiviral Dab1 was detected with an {alpha}-myc antibody. C, fractionation of neuronal lysate (from B) expressing empty vector, wild-type Dab1, K45E, or S114T/F158V mutant constructs. Fractions were analyzed by Western blot for myc (top panel), Akt (middle panel), or LRP (bottom panel). D, Dab1 knockout neurons transduced with native Dab1, the K45E mutant, or empty vector at a higher m.o.i. than in C were fractionated and analyzed by Western blot for Dab1 (top panel), Akt (middle panel), and Synaptophysin (Syn., bottom panel).

 
Preparation of Primary Neurons—All animal experiments were conducted according to the procedures approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Southwestern Medical Center. Primary forebrain neurons were prepared from E15.5–17.5 embryos from timed pregnant wild-type and Dab1 heterozygous mice, or E18–20 rat embryos from timed pregnant Sprague-Dawley rats (Charles River Laboratories) as described (7, 8) and were cultured on poly-D-lysine-coated cell culture plates in a serum-free medium (Neurobasal, Invitrogen), supplemented with 2% B27, 1 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate) at 37 °C in a 5% CO2 environment.

Cellular Fractionation of Primary Neurons—On days 4–6 after culturing, the forebrain neurons were tranduced with the lentiviral particles at m.o.i. of 0.2–0.5. Twenty-four hours after infection the viral supernatant was removed and replaced with conditioned Neurobasal media. Two to 3 days after infection, cellular fractionation was carried out as described (7). In brief, neurons were harvested and lysed by being subjected to a pressure of 500 p.s.i. for 15 min, and forced through a small hole by slowly releasing the pressure. Nuclei were removed by low-speed centrifugation, and the postnuclear supernatants were separated into soluble and particulate fractions by ultracentrifugation. Particulate fractions were re-solubilized, and the protein concentrations of the particulate and soluble fractions were estimated using the Bio-Rad detergent-compatible assay. Equal amounts of protein from each fraction were then analyzed by Western blot with a polyclonal {alpha}-Dab1 antibody (7) or monoclonal {alpha}-Myc antibody (9E10), a polyclonal {alpha}-Akt antibody (Cell Signaling Technology/New England Biolabs), and a polyclonal {alpha}-LRP1 (24) or monoclonal {alpha}-Synaptophysin antibody (SY38, Synaptic Systems).

Immunoprecipitation—On days 4–6 after culturing of the forebrain neurons, the cultures were transduced with the lentiviral particles at m.o.i. of 0.2–0.5. Twenty-four hours after infection the viral supernatant was removed and replaced with conditioned Neurobasal media. Two to 3 days after infection, the neurons were stimulated with partially purified Reelin-conditioned or mock-conditioned media for 15 min at 37 °C and lysed as described (7). The cleared lysates were incubated with 5 µg of a polyclonal rabbit {alpha}-Myc antibody (Bethyl Labs) at 4 °C overnight. This mixture was subsequently incubated with Protein A beads (Sigma) for 2–4 h at 4 °C. The beads were washed 3 times, once with 20 mM Tris buffer, pH 7.5, containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% Triton X-100, protease and phosphatase inhibitors, and twice with the same buffer containing only 0.1% Triton X-100. The beads were resuspended in SDS-PAGE loading buffer, boiled, and analyzed by Western blot using a monoclonal {alpha}-Myc antibody (9E10) as well as a monoclonal {alpha}-phosphotyrosine antibody (4G10, Upstate).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In a previous study (20) we determined the binding affinities of various Dab1 PTB domain mutants for an NPXY-containing peptide from ApoER2, as well as for the phosphoinositide head group from PI 4,5-P2 (20). Mutations that led to loss of binding to one ligand but not the other were introduced into a full-length Dab1 clone to be further characterized in cell-based assays. As we have shown earlier (20), point mutations at either the NPXY or PI binding site (Fig. 1, C and D) selectively eliminate binding at one site but not the other. Mutation of lysine 45 to glutamate (K45E) reduces PI 4,5-P2 binding by at least 300-fold (20), whereas a double mutant of serine 114 to threonine and phenylalanine 158 to valine (S114T/F158V) reduces binding of the NPXY peptide by at least 150-fold (Fig. 2).



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  		FIG. 2.
Isothermal titration calorimetry measurements for ligand binding by the S114T/F158V mutant Dab1 PTB domain. Titrations were performed with 20 µM PTB domain, to which ligand was added incrementally (see "Experimental Procedures"). A, binding of the S114T/F158V mutant PTB domain to PI 4,5-P2. To calculate the dissociation constant, the data were fitted to a one-site binding model with the software program Origin 5.0. B, loss of binding of the S114T/F158V mutant PTB domain to the NPXY peptide. The limit for the dissociation constant was estimated from the total amount of ligand added during the titration. The error measurement given in A is the error in fit. The estimated error in KD upon repeated measurement is ±25% (mean ± S.E.) (20).

 
The K45E mutant (Fig. 1D) retains native affinity for the NPXY peptide ligand but shows negligible binding to PI ligands, and was therefore chosen as a mutant lacking the PI binding ability. To eliminate binding to the NPXY motif, mutations in two important NPXY-interacting residues, serine 114 and phenylalanine 158, were combined to ensure complete loss of binding (Fig. 1C) (19). The S114T/F158V double mutant retains native affinity for PIs (KD = 0.7 ± 0.1 µM), but shows negligible binding to the NPXY-peptide (KD reduced by at least 150–200-fold), as shown by isothermal titration calorimetry (Fig. 2). In addition, the 5F mutant (6), in which tyrosines 185, 198, 200, 220, and 232 are mutated to phenylalanine and that therefore cannot undergo tyrosine phosphorylation, was used as a negative control (Fig. 1A).

Testing of Lentiviral Constructs—Full-length native Dab1 as well as the 5F, K45E, and S114T/F158V mutants containing a carboxyl-terminal 5-Myc tag were subcloned into a lentiviral expression vector derived from LL3.7 (21). To test for Dab1 expression and to show that the mutant Dab1 forms have the potential to undergo tyrosine phosphorylation in response to Reelin, we transiently transfected the different constructs into HEK-293 cells (Fig. 3), either with (+, even numbered lanes) or without (-, odd-numbered lanes) co-transfection with an expression vector containing a Fyn cDNA. This non-receptor tyrosine kinase is the major tyrosine kinase that has been shown to phosphorylate Dab1 in vivo (14, 15). All of the mutants are properly expressed in this heterologous cell system, as shown by detection with a polyclonal {alpha}-Dab1 antibody in HEK-293 cell lysates (upper panel), and all but the 5F mutant (lanes 5 and 6) are phosphorylated, as assessed by Western blotting of the same samples with an {alpha}-phosphotyrosine antibody (lower panel).



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  		FIG. 3.
Expression and tyrosine phosphorylation of Dab1 mutant constructs in 293 cells. The Myc-tagged Dab1 constructs were transfected into 293 cells either with (even-numbered lanes) or without (odd-numbered lanes) co-transfection of a plasmid expressing Fyn tyrosine kinase. The cells were harvested and the cell lysates were analyzed by Western blot for expression of Myc and Phosphotyrosine (pTyr). Top panel, detection of Myc-tagged Disabled-1. Bottom panel, detection of tyrosine-phosphorylated Disabled-1. Vec, empty vector; WT, wild-type Dab1. Dab1 mutants are identified by their amino acid substitutions.

 
Native Dab1, 5F, K45E, and S114T/F158V constructs were then incorporated into lentiviral particles for transduction of primary neuronal cultures. Viral expression was tested by transducing wild-type primary neuronal cultures with each of the different lentiviruses at m.o.i. of 0.2–0.5. Viral expression of Dab1 was detected by blotting with an {alpha}-Myc antibody (Fig. 4, upper panel). All of the viruses expressed comparable amounts of Dab1. Cdk5 (lower panel) served as a loading control.



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  		FIG. 4.
Viral expression of Disabled-1 constructs in primary neurons. Primary neurons from mouse forebrain (day E16.5) were transduced with lentiviral particles expressing different myc-tagged Dab1 mutants (see "Experimental Procedures"). The cells were harvested and the cell lysates were analyzed by Western blot for the presence of myc-tagged protein (top panel). Detection of Cdk5 (Santa Cruz Biotechnology) was used as a control for total protein levels (bottom panel). Vec, empty vector; WT, wild-type Dab1. Dab1 mutants are identified by their amino acid substitutions.

 
Effect of PTB Mutants on Membrane Localization—We then examined the membrane localization of native and mutant forms of Dab1 using a cell fractionation approach (7). Primary neurons were prepared from either Dab1 knockout mouse embryos or from wild-type rat embryos and transduced with the indicated lentiviral constructs at m.o.i. of 0.2–0.5. Forty-eight to 72 h after transduction, the neurons were harvested, homogenized, and separated into soluble (S, cytosolic) and particulate (P, membrane containing) fractions. Membrane fractionations of non-transduced wild-type neurons and knockout neurons transduced with vector alone served as positive and negative controls, respectively. In wild-type neurons, native Dab1 was detected with a polyclonal {alpha}-Dab1 antibody. This same antibody was used to detect virally expressed Dab1 in experiments performed with knockout neurons, whereas an {alpha}-Myc antibody was used for virally expressed Dab1 in wild-type neurons. Detection of Akt was used as a control for a cytoplasmic protein that is present predominantly in the soluble protein fraction (7), whereas LRP1 or Synaptophysin was used as a marker for the membrane containing fraction (7, 25).

Native Dab1 was found exclusively in the particulate fraction of wild-type neurons (7) (Fig. 5A, lane 2). As shown in Fig. 5B myc-tagged wild type (lane 2), K45E (lane 3), and S114T/F158V (lane 4) Dab1 constructs were efficiently expressed by lentivirus-transduced wild-type neurons. Virally expressed wild-type (Fig. 5C, lanes 3 and 4) and S114T/F158V mutant (lanes 7 and 8) Dab1, which both retain PI binding activity, were found predominantly in the particulate, membrane containing fraction in knockout neurons. By contrast, the K45E mutant was present both in the soluble and particulate fractions (lanes 5 and 6, respectively). Infection of neurons at a higher m.o.i. caused a significant accumulation of wild-type Dab1 in the soluble fraction (Fig. 5D, lane 1). The K45E form of Dab1 was almost exclusively soluble at this higher rate of infection. Taken together, these findings suggest that interaction with phosphoinositides, but not with the NPXY motifs, is required for the membrane association of Dab1.

Effect of PTB Mutants on Reelin Signal Transduction—Next, we tested each mutant for its ability to participate in Reelin signaling, i.e. to undergo tyrosine phosphorylation upon Reelin binding to its receptors (4, 8, 14, 26). Wild-type primary mouse or rat neurons were transduced with lentiviral particles expressing the different Dab1 mutants (Fig. 6A, lanes 3–8) or an empty virus (lanes 1 and 2) at an m.o.i. of 0.2–0.5. Two to 3 days after transduction, the neurons were re-fed with either control (mock, M) or Reelin (R) containing medium for 15 min at 37 °C. Cell lysates were immunoprecipitated with an {alpha}-Myc antibody and analyzed by Western blotting with {alpha}-Myc (to monitor protein expression) and {alpha}-Phosphotyrosine antibodies (Fig. 6). Upon Reelin stimulation, native Dab1 is efficiently tyrosine phosphorylated (Fig. 6, A, upper panel, lane 4; B, upper panel, lane 2). As expected, the 5F mutant (Fig. 6A, lanes 5 and 6), which does not contain tyrosine residues, could not be phosphorylated in response to Reelin. The S114T/F158V mutant (lanes 7 and 8) was also resistant to Reelin-induced tyrosine phosphorylation, indicating that high affinity interaction of the Dab1 PTB domain with the NPXY motifs is necessary for signal propagation. Likewise, the K45E mutant was completely resistant to Reelin-induced tyrosine phosphorylation (Fig. 6B, lane 4), although it was expressed at higher levels (bottom panel, lanes 3 and 4) than the wild-type Dab1 (lanes 1 and 2) that served as a control in this experiment. Thus, PI-mediated membrane association of Dab1 is critical for assembly of the Reelin signaling complex at the plasma membrane.



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  		FIG. 6.
Tyrosine phosphorylation of Disabled-1 mutants upon Reelin stimulation. Primary neurons were transduced with lentiviral particles expressing different Myc-tagged Dab1 constructs. The cells were exposed to mock or Reelin-conditioned media and then lysed. The virally expressed Dab1 was immunoprecipitated from the lysate using a polyclonal {alpha}-Myc antibody, and then analyzed by Western blot for levels of phosphotyrosine and Myc. M, mock-conditioned media; R, Reelin-conditioned media. Vec, empty vector; WT, wild-type Dab1. Dab1 mutants are identified by their amino acid substitutions. The empty vector and 5F samples serve as negative controls. A, immunoprecipitated (IP) Western blot to analyze phosphorylation of the S114T/F158V NPXY binding mutant. B, immunoprecipitated Western blot to analyze phosphorylation of the K45E PI binding mutant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Dab1 is as an essential adaptor protein that is required for the relaying of the Reelin signal across the plasma membrane. The amino-terminal PTB domain of Dab1 is critical for mediating the interaction of Dab1 with the Reelin receptors. The same domain also interacts with phospholipids in the cell membrane.

Taken together, the membrane fractionation and phosphorylation experiments we have presented in this paper identify two distinct roles of the Dab1 PTB domain, both of which are required for transduction of a Reelin signal by Dab1: (i) membrane recruitment of the adaptor protein, which requires the PI binding site, and (ii) binding of Dab1 to the ApoER2 tail, which is mediated by interaction with the NPXY motif. The present data suggest that membrane sequestration occurs prior to receptor binding, because NPXY binding mutants have no effect on the partitioning of Dab1 into the membrane fractions, yet both types of mutations eliminate Reelin signal transduction as assessed by tyrosine phosphorylation of Dab1. Thus, we propose a two-step model for ligand binding, where Dab1 first associates with the membrane through binding of PI 4,5-P2, which facilitates the second step of binding to the NPXY motif in the lipoprotein receptor tails by bringing the two binding partners into closer proximity and increasing the effective concentration of Dab1 at the membrane. Simultaneous binding at both sites, followed by Reelin-induced receptor clustering (27) is required for tyrosine phosphorylation of Dab1 and signal propagation (Fig. 7). This model provides a framework for further experimentation to explore the mechanism of these interactions in more detail.



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  		FIG. 7.
A two-step model for ligand binding by the Dab1 PTB domain. The Dab1 PTB domain (A) first associates with PI 4,5-P2 in the cell membrane (B). Association with PI 4,5-P2 increases the local concentration of Dab1 at the membrane and facilitates the interaction with the ApoER2 or very low density lipoprotein receptor cytoplasmic domain (C). The Dab1 PTB domain is associated with both ligands at the cell membrane, and is able to transduce a Reelin signal through phosphorylation in the tyrosine-rich region downstream of the PTB domain (D). TM, transmembrane segment.

 
Interaction of Dab1 with phosphoinositides is necessary for membrane partitioning and Dab1 phosphorylation (Figs. 5 and 6). This suggests that loss of membrane binding prevents effective association of Dab1 with ApoER2 and thus does not allow Dab1 phosphorylation to occur. However, it is also possible that PI binding by Dab1 has an additional effect on Dab1 function. For instance, the incorporation of two binding sites on a single domain in a certain orientation may be critical for the proper orientation and presentation of Dab1 at the plasma membrane. Thus, PI binding by the Dab1 PTB domain may facilitate interaction with the ApoER2 cytoplasmic domain, and possibly also with the regulatory 85-kDa subunit of PI 3-kinase (7) by ensuring that the Dab1 PTB domain is properly integrated into the membrane signaling complex. Further experiments will be necessary to distinguish whether PI binding serves only for membrane recruitment or whether it also serves an additional orientation function. For example, a Dab1 mutant that is unable to bind PIs, but instead contains a myristoylation signal could be designed to test whether membrane recruitment by lipid modification is able to replace PI binding and rescue Reelin signal transduction. Failure to do so would suggest that PI binding has an additional role beyond that of recruitment.

A precedent for the importance of domain orientation at the membrane is found in the Dbs protein, a member of the Dbl family of guanine nucleotide exchange factors that contain tandem Dbl homology and PH domains. The Dbl homology domain is necessary for interaction with GTPases and for proper nucleotide exchange, and is always found in tandem with a carboxyl-terminal PH domain. The orientation of these two domains relative to each other is invariant. Studies on the PH domain of the Dbs protein show that although mutations in the PI binding site of the PH domain do not affect membrane localization, they do affect the ability of the Dbs protein to activate the RhoA GTPase (28). This suggests that PI binding by the Dbs PH domain has another function, and may serve to orient Dbl homology with respect to the PH domain to facilitate interaction with Rho GTPases.

Such an orientation requirement is not limited to proteins containing PH or PTB domains. The PTEN tumor suppressor contains a phosphatase domain as well as a C2 domain, which regulates association with the membrane. These two domains have an extensive interface involving highly conserved amino acids, and mutations that disrupt this interface have been found in human tumors. They also greatly reduce PTEN phosphatase activity (29). This implies that the orientation between the phosphatase and C2 domains is critical for productive substrate binding by the phosphatase domain.

Because PI 4,5-P2 is thought to be abundant in caveolae or lipid rafts, PI binding by Dab1 may also function to localize this protein to this particular region of the cell membrane. There is evidence that other members of the Reelin signal transduction machinery, including ApoER2 (30) and Src family members, are localized to lipid rafts. The intracellular area surrounding the cell membrane is occupied by numerous proteins, thereby decreasing the opportunity for two proteins to come into contact with one another simply through cellular diffusion. PI binding by the Dab1 PTB domain and sequestration of the protein to a subcompartment of the two-dimensional plasma membrane thus greatly increases the probability for a productive encounter between Dab1 and the NPXY motifs in the intracellular domains of the Reelin receptors, as compared with the fluidic three-dimensional space of the cell.

Whereas other PTB domains are able to bind to PIs in vitro, little is known about the physiological role of PI binding by PTB domains in vivo or in cells. Although the Shc PTB domain also has been reported to bind to PIs, this interaction occurs at a site that competes with the binding of the phosphotyrosine containing peptide sequence, and at a much lower affinity than would be necessary for membrane recruitment (31). Indeed, the bulk of total cellular Shc protein is not associated with the membrane in fractionation experiments (32). However, Shc mutants that are unable to interact with PIs in vitro are also unable to undergo interleukin-3-dependent phosphorylation in cell culture experiments (32). Whether this effect is related to loss of PI binding is unclear, as the exact binding site for PIs on the Shc PTB domain has not been characterized.

The ARH and Dab2 PTB domains, which are the closest homologues of Dab1, have also been shown to bind PIs in vitro (33, 34). The structure of the Dab2 PTB domain reveals a similar patch of basic residues that most likely also mediates PI binding (18), and a structural model of the ARH PTB domain also shows the characteristic grouping of basic residues that is the signature of a PI binding patch (19). In contrast to Dab1, which mediates transduction of a signal across the cellular membrane, ARH and Dab2 function as adaptors that connect lipoprotein receptors, and possibly other membrane proteins, to the endocytosis machinery through clathrin binding motifs in their carboxyl termini (33–35). Thus, the type of dual-ligand binding PTB domain represented by Dab1 is likely to be used as a structural unit by multiple types of adaptor proteins to mediate a wide range of cellular processes.

The results we have presented here have revealed an important role for PI binding by the Dab1 PTB domain in the transduction of the Reelin signal. The definitive physiological importance of the PI binding site will ultimately have to be investigated in knock-in mice in which the K45E mutation has been introduced into the endogenous Dab1 gene. A recent study by Herrick and Cooper (36) has used this technique to investigate the effect of the single F158V NPXY binding mutation on brain development. Mice hemizygous for this mutation present as a hypomorphic phenocopy of the Dab1 knockout (36). This milder phenotype is most likely because of the fact that the F158V mutation alone, without the accompanying S114T mutation we have used in our studies, results only in an ~10-fold reduction of affinity of the PTB domain for the NPXY peptide in vitro (20), which may still allow sufficient Reelin signal propagation. Incorporation of mutations leading to more dramatic loss of either peptide or PI binding in vivo are now needed to fully define the physiological importance of either binding site for brain development and synapse function (35).


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL20948, HL63762, NS43408, and HL61001, the Wolfgang-Paul program of the Humboldt Foundation, and the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§§ Established Investigator of the American Heart Association and a Pew Scholar in the Biomedical Sciences. Back

§ Present address: Max-Planck-Institute for Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany. To whom correspondence may be addressed. E-mail: peggy.stolt{at}mpibpfrankfurt.mpg.de. {ddagger}{ddagger} To whom correspondence may be addressed. E-mail: hans.bock{at}zfn.uni-freiburg.de.

1 The abbreviations used are: ApoER2, apolipoprotein E-receptor 2; Dab1, Disabled-1; EGFP, enhanced green fluorescent protein; LRP, low density lipoprotein receptor-related protein 1; m.o.i., multiplicity of infection; NPXY, Asn-Pro-(any amino acid)-Tyr; PH, pleckstrin homology; PI, phosphoinositide; PI 4,5-P2, phosphoinositide 4,5-bisphosphate; PTB, phosphotyrosine binding; CMV, cytomegalovirus; PIPES, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Wen-Ling Niu, Hui-chuan Reyna, Megan Davenport, and Jill Fairless for excellent technical assistance, and Uwe Beffert for many helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ogawa, M., Miyata, T., Nakajima, K., Yagyu, K., Seike, M., Ikenaka, K., Yamamoto, H., and Mikoshiba, K. (1995) Neuron 14, 889-912[CrossRef][Medline] [Order article via Infotrieve]
  2. Hirotsune, S., Takahara, T., Sasaki, N., Hirose, K., Yoshiki, A., Ohashi, T., Kusakabe, M., Murakami, Y., Muramatsu, M., Watanabe, S., Nakao, K., Katsuki, M., and Hayashizaki, Y. (1995) Nat. Genet. 10, 77-83[CrossRef][Medline] [Order article via Infotrieve]
  3. D'Arcangelo, G., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K., and Curran, T. (1997) J. Neurosci. 17, 23-31[Abstract/Free Full Text]
  4. Hiesberger, T., Trommsdorff, M., Howell, B. W., Goffinet, A., Mumby, M. C., Cooper, J. A., and Herz, J. (1999) Neuron 24, 481-489[CrossRef][Medline] [Order article via Infotrieve]
  5. D'Arcangelo, G., Homayouni, R., Keshvara, L., Rice, D. S., Sheldon, M., and Curran, T. (1999) Neuron 24, 471-479[CrossRef][Medline] [Order article via Infotrieve]
  6. Howell, B. W., Lanier, L. M., Frank, R., Gertler, F. B., and Cooper, J. A. (1999) Mol. Cell. Biol. 19, 5179-5188[Abstract/Free Full Text]
  7. Bock, H. H., Jossin, Y., Liu, P., Forster, E., May, P., Goffinet, A., and Herz, J. (2003) J. Biol. Chem. 278, 38772-38779[Abstract/Free Full Text]
  8. Beffert, U., Morfini, G., Bock, H. H., Reyna, H., Brady, S. T., and Herz, J. (2002) J. Biol. Chem. 277, 49958-49964[Abstract/Free Full Text]
  9. Howell, B. W., Hawkes, R., Soriano, P., and Cooper, J. A. (1997) Nature 389, 733-737[CrossRef][Medline] [Order article via Infotrieve]
  10. Howell, B. W., Gertler, F. B., and Cooper, J. A. (1997) EMBO J. 16, 121-132[CrossRef][Medline] [Order article via Infotrieve]
  11. Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., Hammer, R. E., Richardson, J. A., and Herz, J. (1999) Cell 97, 689-701[CrossRef][Medline] [Order article via Infotrieve]
  12. Howell, B. W., Herrick, T. M., Hildebrand, J. D., Zhang, Y., and Cooper, J. A. (2000) Curr. Biol. 10, 877-885[CrossRef][Medline] [Order article via Infotrieve]
  13. Keshvara, L., Benhayon, D., Magdaleno, S., and Curran, T. (2001) J. Biol. Chem. 276, 16008-16014[Abstract/Free Full Text]
  14. Bock, H. H., and Herz, J. (2003) Curr. Biol. 13, 18-26[CrossRef][Medline] [Order article via Infotrieve]
  15. Arnaud, L., Ballif, B. A., Forster, E., and Cooper, J. A. (2003) Curr. Biol. 8, 9-17
  16. Sanada, K., Gupta, R., and Tsai, L. H. (2004) Neuron 42, 197-211[CrossRef][Medline] [Order article via Infotrieve]
  17. Lemmon, M. A., and Ferguson, K. M. (2000) Biochem. J. 350, 1-18[CrossRef][Medline] [Order article via Infotrieve]
  18. Yun, M., Keshvara, L., Park, C.-G., Zhang, Y.-M., Dickerson, J. B., Zheng, J., Rock, C. O., Curran, T., and Park, H.-W. (2003) J. Biol. Chem. 278, 36572-36581[Abstract/Free Full Text]
  19. Stolt, P. C., Jeon, H., Song, H. K., Herz, J., Eck, M. J., and Blacklow, S. C. (2003) Structure 11, 569-579[Medline] [Order article via Infotrieve]
  20. Stolt, P. C., Vardar, D., and Blacklow, S. C. (2004) Biochemistry 43, 10979-10987[CrossRef][Medline] [Order article via Infotrieve]
  21. Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., Sievers, C., Yang, L., Kopinja, J., Rooney, D. L., Ihrig, M. M., McManus, M. T., Gertler, F. B., Scott, M. L., and Van Parijs, L. (2003) Nat. Genet. 33, 401-406[CrossRef][Medline] [Order article via Infotrieve]
  22. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono, D. (1996) Science 272, 263-267[Abstract]
  23. Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., and Naldini, L. (1998) J. Virol. 72, 8463-8471[Abstract/Free Full Text]
  24. May, P., Rohlmann, A., Bock, H. H., Zurhove, K., Marth, J. D., Schomburg, E. D., Noebels, J. L., Beffert, U., Sweatt, J. D., Weeber, E. J., and Herz, J. (2004) Mol. Cell. Biol. 24, 8872-8883[Abstract/Free Full Text]
  25. Wiedenmann, B., and Franke, W. W. (1985) Cell 41, 1017-1028[CrossRef][Medline] [Order article via Infotrieve]
  26. Howell, B. W., Herrick, T. M., and Cooper, J. A. (1999) Genes Dev. 13, 643-648[Abstract/Free Full Text]
  27. Strasser, V., Fasching, D., Hauser, C., Mayer, H., Bock, H. H., Hiesberger, T., Herz, J., Weeber, E. J., Sweatt, J. D., Pramatarova, A., Howell, B., Schneider, W. J., and Nimpf, J. (2004) Mol. Cell. Biol. 24, 1378-1386[Abstract/Free Full Text]
  28. Rossman, K. L., Cheng, L., Mahon, G. M., Rojas, R., Snyder, J. T., Whitehead, I. P., and Sondek, J. (2003) J. Biol. Chem. 278, 18393-18400[Abstract/Free Full Text]
  29. Lee, J.-O., Yang, H., Gergescu, M.-M., di Cristofano, A., Maehama, T., Shi, Y., Dixon, J. E., Pandolfi, P., and Pavletich, N. P. (1999) Cell 99, 323-334[CrossRef][Medline] [Order article via Infotrieve]
  30. Riddell, D. R., Sun, X. M., Stannard, A. K., Soutar, A. K., and Owen, J. S. (2001) J. Lipid Res. 42, 998-1002[Abstract/Free Full Text]
  31. Zhou, M. M., Ravichandran, K. S., Olejniczak, E. F., Petros, A. M., Meadows, R. P., Sattler, M., Harlan, J. E., Wade, W. S., Burakoff, S. J., and Fesik, S. W. (1995) Nature 378, 584-592[CrossRef][Medline] [Order article via Infotrieve]
  32. Ravichandran, K. S., Zhou, M. M., Pratt, J. C., Harlan, J. E., Walk, S. F., Fesik, S. W., and Burakoff, S. J. (1997) Mol. Cell. Biol. 17, 5540-5549[Abstract]
  33. Mishra, S. K., Keyel, P. A., Hawryluk, M. J., Agostinelli, N. R., Watkins, S. C., and Traub, L. M. (2002) EMBO J. 21, 4915-4926[CrossRef][Medline] [Order article via Infotrieve]
  34. Mishra, S. K., Watkins, S. C., and Traub, L. M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 16099-16104[Abstract/Free Full Text]
  35. He, G., Gupta, S., Yi, M., Michaely, P., Hobbs, H. H., and Cohen, J. C. (2002) J. Biol. Chem. 277, 44044-44049[Abstract/Free Full Text]
  36. Herrick, T. M., and Cooper, J. A. (2004) Brain Res. Mol. Brain Res. 126, 121-128[Medline] [Order article via Infotrieve]

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