Structural and functional basis of a role for CRKL in a fibroblast growth factor 8-induced feed-forward loop

Abstract

The adapter protein CRKL is required for the normal development of multiple tissues that rely on fibroblast growth factor 8 (FGF8). The precise role of CRKL in receptor signaling has been unclear, however. To address this issue, we first modeled the three-dimensional structure of CRKL by molecular dynamics. By taking advantage of structural simulations, we performed in silico analysis of the interactions of the autophosphorylation sites of FGR receptor 1 (FGFR1) with the SH2 domain of CRKL or a highly related protein, CRK. As predicted by simulations, we confirm the specific physical interaction of phosphorylated Y463 (pY463) in FGFR1 with the CRKL SH2 domain at an affinity approximately 30-fold stronger than that of CRK. We also provide evidence that interactions outside of the core YXXP motif have a significant impact on physical association, which is consistent with predictions from molecular-dynamics simulations. Furthermore, we identify CRKL as an essential component of an FGF8-induced feed-forward loop permissive for efficient activation of the mitogen-activated protein kinase Erk1/2, as well as FGF8-induced anchorage-independent cell growth, using Crkl-deficient cells or a pY463 synthetic peptide. Although many cells generally require cell-matrix adhesion, our results demonstrate that CRKL permits cells to bypass the strict need for adhesion in response to FGF8 through direct interaction with receptor.

FIG. 1.

Full-length structures of CRK and CRKL predicted by MD simulation. (A) Structure of full-length CRK (isoform a) with or without phosphorylation at Y221. (B) Structure of CRKL with or without phosphorylation at Y207. These structures were obtained by MD simulations as the average structure of 7- to 10-ns simulations (shown in ribbon representations). The SH2, SH3n, and SH3c domains are highlighted by blue, pink, and slate, respectively. The linker sequences between domains are shown in green. The internal phosphotyrosine residue (pY221 and pY207 in CRK and CRKL, respectively) is shown in magenta; the tyrosine residues without phosphorylation are shown in yellow.

FIG. 2.

Structural basis of interaction between CRKL SH2 domain and pY peptide. (Structural information is available in the supplemental material.) (A) Electrostatic interaction between pY463 in the peptide sequence and two arginine residues in the SH2 domain. pY463 is shown in magenta. SH2 domains are shown in ribbon representations in green, except that two arginine residues interacting with the phosphotyrosine are shown in a stick model. The number shown between the two lines is the angle formed by the central carbon atom in the guanidinium group in the two arginine residues and the phosphorus atom in the phosphate group in pY463 to illustrate qualitative differences in the way that CRKL and CRK SH2 domains interact with the phosphotyrosine in the peptide. (B) Interaction of charged residues between the SH2 domain and FGFR1 peptide sequence. The SH2 domain of CRK and CRKL are colored in green in a ribbon representation. The backbone of the phosphorylated Y463 peptide sequence is shown in red. Charged residues in the peptide sequence and SH2 domain are shown in stick or space fill models, respectively. The phosphate group in pY463 is shown in red. Negatively and positively charged amino acids are shown in yellow or light blue, respectively. (C) Summary table for the distance between two atoms involved in electrostatic interactions. (D) Hydrophobic interactions between the SH2 domain and FGFR1 peptide sequence. The backbone of the pY peptide is shown in red. Hydrophobic residues in the peptide sequence are shown in stick models; pY463 in yellow and the others in white. Hydrophobic residues along the peptide binding area in the SH2 domain are shown in cyan in the surface model. Hydrophobic pockets are highlighted by a circle in magenta.

FIG. 3.

Saturation binding experiments for interactions between the SH2 domain of CRKL or CRK and phosphotyrosyl peptides. (A) Representative plots of saturation binding data between the SH2 domain of CRKL or CRK and a peptide corresponding to FGFR1 pY463 (pYELP peptide) or a modified pYELP peptide (pYELP-AA), in which R470/475 are replaced with alanine residues. (B) Representative plots of saturation binding data between CRKL or CRK SH2 domain and a peptide corresponding to BCAR1 pY287 and pY362 peptides. Experiments were carried out using Cy3-labeled SH2 domains after cleavage of GST by thrombin. The x axis shows the concentration of Cy3-labeled SH2 domain, and the y axis shows the arbitrary fluorescent unit (FU) that corresponds to the amount of bound protein. The K_D values are shown below the plots. The 95% confidence interval is shown in parentheses.

FIG. 4.

The physical association of CRKL to Fgf8-stimulated FGFR1 depends on Y463. (A) Coimmunoprecipitation of CRKL and FGFR1. Human embryonic kidney 293 cells were transfected transiently with expression vectors of wild-type (wt) or mutant FGFR1 lacking Y463 (Y463F). Suspension culture was stimulated with 10 ng of Fgf8/ml for 10 min, and then cell lysates were prepared. Fgfr1 was immunoprecipitated (IP: FGFR1). Coimmunoprecipitation with CRKL or CRK was assessed by immunoblotting. The leftmost lane includes 10 μg of total protein in lysate as a control in order to estimate the amount of associated CRKL or CRK protein relative to the total amount in lysates. In parallel experiments, cell lysates (lysates) were evaluated by SDS-PAGE and immunoblotted with the antibody indicated. (B) pYELP peptide inhibits association of Crkl with Fgfr1. A suspension culture of wild-type MEFs was stimulated with 10 ng of Fgf8/ml for 10 min, and then cell lysates were prepared. Peptide was added to cell lysates at different concentrations, and Fgfr1 was immunoprecipitated. Coimmunoprecipitation with Crkl was assessed by immunoblotting. (C) pYELP peptide inhibits in vivo association of Crkl with Fgf8-stimulated Fgfr1. Suspension cultures of wild-type MEFs were pretreated with either pYELP or YELP peptide mixed with the carrier peptide PEP-1 and then stimulated with 10 ng of Fgf8/ml for 10 min. Cell lysates were prepared, and Fgfr1 or Fgfr2 was immunoprecipitated. Coimmunoprecipitation of Fgfr1 with Crkl was assessed by immunoblotting.

FIG. 5.

Crkl is essential for Fgf8-induced activation of Rac1 and Cdc42, but not Ras. (A) Pulldown assays for GTP-bound small G proteins. Wild-type MEFs were stimulated with either Fgf8 or EGF, while cells were kept in suspension. Cell lysates were prepared after 10 min of stimulation. The levels of total Ras, Rac1, and Cdc42 are shown in the lower panel for each GTP-bound G protein pulldown. (B) Pulldown assays for GTP-bound small G proteins after 10 min of stimulation with 10 ng of Fgf8/ml in suspension culture of MEFs isolated from wild-type or Crkl^−/− embryos. An additional control was prepared by introducing a CRKL transgene into Crkl^−/− MEFs (Crkl^−/− + CRKL). A pulldown assay of GTP-bound small G-proteins was carried out as described above. (C) Coimmunoprecipitation of Fgfr1 with Dock1. Cell lysates were prepared as described above to immunoprecipitate Fgfr1. Dock1 protein was probed in the immunoprecipitate by immunoblotting.

FIG. 6.

Crkl is essential for Fgf8-induced activation of the cascades of serine/threonine kinases. (A) In vitro kinase assays for Pak, Raf1, and Mek1. Suspension culture of MEFs was stimulated with 10 ng of Fgf8/ml for 10 min, and each protein was immunoprecipitated from cell lysates for in vitro kinase assays. (B) Immunoblot analysis for Fgf8-induced site specific phosphorylation of Pak, Raf1, Mek1, and Erk1/2. Cell lysates were prepared as described above, and phospho-specific antibodies were used to probe phosphorylation events. Total protein levels of each protein are shown in the lower panel for each protein.

FIG. 7.

pYELP inhibits cellular responses to Fgf8. (A) pYELP peptide inhibits Fgf8-induced activation of Rac1 and Cdc42, but not Ras. A suspension culture of wild-type MEFs was pretreated with either pYELP or YELP peptide mixed with the carrier peptide PEP-1, and then cells were stimulated with 10 ng of Fgf8/ml for 10 min. Cell lysates were prepared, and pulldown assays were performed. (B) pYELP peptide inhibits Fgf8-induced Erk1/2 activation. Peptide and Fgf8 treatment was done as described above. Cell lysates were analyzed by immunoblots for phospho-Erk. (C) CRKL is essential for anchorage-independent cell growth induced by Fgf8. Colony-forming assays were performed using MEFs as described in Materials and Methods. Photographs of the colonies are shown.

FIG. 8.

Model of the network that activates the MAP kinase by FGF8. The figure illustrates a role for the feed-forward loop mediated by interaction of CRKL and FGFR1 in organizing the output through the MAP kinase ERK1/2. Note that RAF1 and MEK1 function as tandem AND-gates that integrate signals effectively from FGFR1. Although cell-matrix adhesion can also participate in this network (shaded in gray), cells stimulated by FGF8 can escape from this requirement. This is a simplified model that does not show additional regulatory pathways mediated by other signaling modifiers.