QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 17 August 2004
doi: 10.1242/jcs.01320

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, K. Articles by Howell, B. W. Search for Related Content PubMed PubMed Citation Articles by Chen, K. Articles by Howell, B. W. Social Bookmarking                         What's this?

Interaction between Dab1 and CrkII is promoted by Reelin signaling

¹ Neurogenetics Branch, NINDS/NIH, 10 Center Drive, Bethesda, MD 20892-1250, USA
² Molecular Virology and Neurogenetics Section, NINDS/NIH, 10 Center Drive, Bethesda, MD 20892-1250, USA

View larger version (35K): [in a new window]   Fig. 1. Isolation and identification of phosphotyrosine-dependent Dab1-binding proteins from embryonic mouse brain. (A) Two protein bands are observed to bind specifically to phosphorylated GST-Dab1 by silver staining samples resolved by SDS-PAGE (arrows). Embryonic brain lysates were incubated with immobilized tyrosine-phosphorylated GST-Dab1 (pY, lane 1) or unphosphorylated Dab1 (Y, lane 2) and bound proteins were eluted with 100 mM phenyl phosphate solution. The band at approximately 180 kDa was more distinct in the preparative gels used for protein identification. (B) Dock1, associated with tyrosine-phosphorylated GST-Dab1 (lane 1), but not unphosphorylated GST-Dab1 (lane2), was detected by western blotting samples eluted with sample buffer. (C) Crk was observed to interact with tyrosine-phosphorylated GST-Dab1 (lane 1) but not unphosphorylated GST-Dab1 (lane 2) by western blotting samples eluted with sample buffer.

View larger version (29K): [in a new window]   Fig. 2. CrkII mediates the Dock1 interaction with Dab1 and requires Dab1 Y220 or Y232 for binding. (A) Dock1 (lanes 1-3, upper panel) and CrkII (lanes 4-6, upper panel) were detected by western blotting lysates from HEK293T cells transfected with Dock1 (lanes 2,3), and/or CrkII (lanes 1,3). Dock1 only associated with the immobilized tyrosine-phosphorylated GST-Dab1 in the presence of overexpressed CrkII (lane 3, lower panel) and not in its absence (lane 2, lower panel). In contrast, Crk II associated with GST-Dab1 in both the absence (lane 4, lower panel) and presence (lane 6, lower panel) of Dock1. (B) Expression of Dab1 wild-type (lanes 1,3, upper panel), the indicated Dab1 tyrosine to phenylalanine mutants (lanes 4-7, upper panel) and CrkII (lanes 9-14, upper panel) was detected in total cell lysates of transfected HEK293T cells, which were cotransfected with the activated Src527F mutant. Co-immunoprecipitation of Dab1 and CrkII was detected by immunoprecipitation with anti-CrkII (lanes 1-7, lower panel) or anti-Dab1 (lanes 8-14, lower panel) and western blotting with anti-Dab1 (lanes 1-7, lower panel) or anti-CrkII antibodies (lanes 8-14, lower panel). Wild-type Dab1 (lanes 3,10, lower panel) and single-site substituted Dab1 (lanes 4-6 and 11-13, lower panel), but not the Dab1 Y220F-Y232F double mutant (lanes 7,14, lower panel) supported co-immunoprecipitation with CrkII. Dab1 molecules that are phosphorylated at Y232 have reduced electrophoretic mobility as compared with unphosphorylated Dab1 or Dab1 with phenylalanine substitutions at this position (compare lanes 1 and 3-5 with 6 and 7, lower panel) (see also Howell et al., 2000). This figure shows representative results obtained in three independent experiments.

View larger version (62K): [in a new window]   Fig. 3. CrkII forms a Reelin-promoted complex with Dab1 in embryonic forebrain neurons. Total cell lysates (TCL) were collected from cultured embryonic forebrain neurons which were unstimulated (0; lanes 1,4), stimulated with control conditioned media (CCM; lanes 2,5), or stimulated with Reelin conditioned media (RCM; lanes 3,6) for 15 minutes. Western blotting these samples with anti-phosphotyrosine antibody (pY; 4G10) shows the induction of the tyrosine phosphorylation of an 80 kDa protein, which corresponds to Dab1 (lanes 1-3, upper panel), whereas the Dab1 protein levels are relatively constant between samples (lanes 1-3, lower panel). Equal levels of CrkII are detected in the anti-CrkII immunoprecipitates from these cell lysates (lanes 4-6, upper panel); however, increased co-immunoprecipitation of Dab1 was detected from lysates of Reelin-treated samples (compare lanes 4-6, lower panel). Approximately 100% of CrkII was immunoprecipitated under the assay conditions, and 5% of total Dab1 was detected in the co-immunoprecipitation (not shown).

View larger version (10K): [in a new window]   Fig. 4. Expression of Dab1RFP, but not the Dab1RFP-5F mutant, reduced the mobility of NBT-II cells plated on collagen. (A) The trajectory of Dab1RFP (red traces) was reduced as compared with GFP-expressing cells (green traces) that were imaged in the same experiment every 6 minutes over a period of 8 hours by time-lapse fluorescence microscopy. (B) The trajectory of Dab1RFP-5F (red traces) was comparable to GFP-expressing cells (green traces). (C) Co-expression of CrkIIGFP with Dab1RFP (purple) restored the ability to migrate on collagen to many of the co-expressing cells, and in the same experiment cells expressing Dab1RFP alone (red) showed little movement (Bar, 60 µm). The cell tracings are a composite of three independent experiments in which similar results were obtained.

View larger version (44K): [in a new window]   Fig. 5. The expression of Dab1RFP alters the morphology and profile of filamentous actin in NBT-II cells plated on collagen. (A) The filamentous actin distribution detected by FITC-phalloidin (green) was polarized and features of migrating cells such as lamellipodia, filopodia and stress fibers were apparent in RFP-expressing (red) NBT-II cells. This was comparable to untransfected cells (not shown). (B) Dab1RFP expression (red) resulted in a morphological change in the cell and loss of lamellipodia and stress fibers, but filopodia were apparent. (C) Expression of Dab1RFP-5F (red) did not lead to the loss of lamellipodia or stress fibers (Bar, 10 µm). Nuclei were detected by Dapi staining (blue). Arrowhead, lamellipodia; arrow, stress fibers. The cells shown are representative of the majority of cells from each transfection.

View larger version (150K): [in a new window]   Fig. 6. Expression of Dab1RFP in NBT-II cells alters CrkII distribution but does not alter phosphorylation of Fak at Y397. (A) Endogenous CrkII (green) was observed to co-localize (yellow areas) with Dab1RFP (red) at the cell membrane of transfected NBT-II cells. (B) In cells expressing Dab1RFP-5F (red) the CrkII signal was not observed to overlap. (C) In cells expressing Dab1RFP, Fak phosphorylated at Y397 (green) was observed in the plane closest to the coverglass. (D) Dab1RFP-5F-transfected cells also expressed phosphorylated Fak.

View larger version (169K): [in a new window]   Fig. 7. Reduced gene dosage of the Drosophila Dock1 homolog, mbc, partially rescues the rough eye phenotype caused by expression of Dab1RFP. (A) The compound eyes of Drosophila with the genotype UAS-RFP/GMR-GAL4 appeared normal as visualized by scanning electron microscopy. (B) Expression of tyrosine-phosphorylated Dab1RFP in Drosophila with the genotype UAS-Dab1RFP/+;GMR-GAL4/+ led to the development of a rough eye morphology characterized by fusions of ommatidia and loss of linearity of the rows of the ommatidial facets (Pramatarova et al., 2003). (C) Heterozygous mutation of mbc partially rescued the developmental phenotype caused by Dab1RFP expression. Drosophila with the genotype UAS-Dab1RFP/+; GMR-GAL4/+; mbc/+ had fewer ommatidial fusions, and the rows of ommatidia are more linear. Magnification: Ai-Ci, 200x (Bar, 100 µm); Aii-Cii, 800x.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter