Differential interaction of the Pafah1b alpha subunits with the Reelin transducer Dab1

Abstract

The Reelin signaling pathway controls radial neuronal migration and maturation in the developing brain. The platelet activating factor (PAF) acetyl hydrolase 1b (Pafah1b) complex is also involved in multiple aspects of brain development. We previously showed that the Reelin pathway and the Pafah1b complex interact genetically and biochemically. Lis1, the regulatory subunit of Pafah1b interacts with phosphoDab1, an essential mediator of Reelin signaling. Compound mutants carrying mutations in both, the Reelin pathway and Lis1 exhibit hydrocephalus, a phenotype that is suppressed by mutations in the gene encoding the Alpha2 subunit of Pafah1b. This subunit, like the Alpha1 catalytic subunit of Pafah1b also binds the Reelin receptor VLDLR. Here we investigated the molecular interactions of the Pafah1b catalytic subunits with Dab1. We found that Alpha2 coprecipitates with Dab1 from brain extracts of normal and reeler mutant mice lacking Reelin, and from cell-free extracts containing normal or a phosphorylation mutant form of Dab1, suggesting that Dab1 phosphorylation is not necessary for binding to Alpha2. This interaction is specific for Alpha2 and not Alpha1, and depends on a unique tyrosine residue of Alpha2. Biochemical assays using mutant mice lacking Alpha2 further demonstrated that this subunit is not required for Reelin-induced Dab1 phosphorylation. However, increasing amounts of Alpha2 in a cell-free system disrupted the formation of Dab1-Lis1 complexes without affecting the association of Dab1 with VLDLR. Our data suggest that the Alpha2 subunit may play a modulatory role in the formation of protein complexes that affect brain development and hydrocephalus.

Figure 1. Alpha2 binds phosphorylated and unphosphorylated Dab1

A, Alpha 2 binds Dab1 in the embryonic brain. Proteins were extracted from the brain of littermates that were wild type, heterozygous or homozygous null for the Pafah1b2 gene, immunoprecipitated with Alpha2 antibodies and probed with Dab1 antibodies (IP, upper panel). The brain lysates were also directly probed with Dab1 antibodies (lower panel) to ensure that similar amounts of proteins were present in each sample. B, Alpha 2 binds Dab1 in normal and reeler brain. Proteins were extracted from the brain of littermates that were wild type, heterozygous or homozygous null for the Reln gene, immunoprecipitated with no antibodies or with Alpha2 antibodies and probed with Dab1 antibodies (IP, upper panel). The brain lysates were also probed with Dab1 (middle panel) or Alpha2 (lower panel) antibodies. Elevated levels of Dab1 protein are present in the homozygous Reln mutant. C, FLAG-tagged subunits of Pafah1b and HA-tagged Dab1 proteins were expressed in vitro using a cell-free-system. Proteins were incubated with or without recombinant Src kinase and immunoprecipitated with FLAG antibodies. The blot (IP) was probed with HA antibodies to detect Dab1 proteins (upper panel). Normal Dab1 interacted with Lis1 only in the presence of Src, whereas normal and mutated Dab1 interacted with Alpha2 with or without the kinase. In vitro translated proteins (TnT) were probed with HA or FLAG antibodies to ensure similar levels of expression (lower panels).

Figure 2. The unique Y56 residue of Alpha2 is required for binding to Dab1

A, Alpha1 does not bind Dab1 in the embryonic brain. Proteins were extracted from wild type embryonic mouse brain and immunoprecipitated with Alpha1, Alpha2, Lis1 or control Myc antibodies and probed with Dab1 antibodies (IP). Dab1 coprecipitated only with Alpha2 or Lis1. B, Amino acid sequence alignment of a highly conserved N terminal region of mouse Alpha1 and Alpha2 proteins containing residue 56 (arrow). C, FLAG-tagged Lis1, GFP-tagged Alpha1 or Alpha2 and HA-tagged Dab1 were coexpressed in COS7 cells. Proteins were immunoprecipitated with the indicated antibodies and probed with GFP antibodies to detect Alpha1 or Alpha2 (IP). The lysates were also directly probed with GFP antibodies to ensure that Alpha1 or Alpha2 proteins were expressed at similar levels. Mutant Alpha2(Y56C), like Alpha1, cannot bind Dab1.

Figure 3. Pafah1b2 mutations do not inhibit Reelin-induced Dab1 phosphorylation

A. Cortical neuron cultures obtained from mice of the indicated Pafah1b2 genotype were treated with Reelin (R) or a mock control medium (M). B. Cortical neuron cultures obtained from mice of the indicated Pafah1b2 and Reln genotypes were treated with Reelin (R) or a mock medium (M). Phospho-Dab1 levels are shown in the top panels. Total Dab1 protein levels are shown in the bottom panels. Reelin induced Dab1 phosphorylation in all samples analyzed.

Figure 4. Alpha2 competes with Lis1 for binding to Dab1 but does not competes with Dab1 for binding to VLDLR

Competition assays in cell-free extracts containing increasing amounts of Alpha2. A. 50μl of in vitro produced Dab1 were incubated with 50 μl of in vitro produced Lis1 and different amount (0, 5, 10, 25, 50 μl) of in vitro produced Alpha2. Proteins were immunoprecipitated with Dab1 antibodies, detected by autoradiography, and normalized to the amount of precipitated Dab1. Histograms show the ratio of Lis1/Dab1 (Grey) and Alpha2/Dab1 (black). B. 50μl of in vitro produced VLDLR were incubated with 50 μl of Dab1 and different amount (0, 5, 10, 25, 50 μl) of Alpha2. Proteins were immunoprecipitated with VLDLR antibodies and normalized to the amount of precipitated receptor. Histograms show the ratio of Dab1/VLDLR (black) and Alpha2/VLDLR (grey).

Figure 5. Putative Lis1- and Dab1-containing protein complexes at different developmental ages

At prenatal agesthe Pafah1b complex consists mostly of Alpha1 dimers and Lis1. Dab1 binds VLDLR, and is heavily phosphorylated by SFKs in response to Reelin. Dab1 also binds ApoER2 (not shown). PhosphoDab1 binds Lis1 in addition to other signaling molecules (not shown for simplicity). VLDLR, but not ApoER2, also binds Alpha1. At perinatal ages, as Alpha1 levels decrease and Alpha2 levels increase new complexes form. The Pafah1b complex becomes heterotrimeric, and Alpha2 competes and displaced Alpha1 from VLDLR. Levels of Dab1 phosphorylation drop and Alpha2 binds Dab1 instead of Lis1. At postnatal ages the Pafah1b complex contains mostly Alpha2 and Lis1. A multiprotein VLDLR-Alpha2-Dab1 complex may form.