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 ∼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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank M. Mohammadi for reagents; M. R. Rosner for sharing protocols for in vitro kinase assays; W.-J. Rhee for peptide synthesis and purification; and B. Liu, P. D. Nash, R. B. Jones, and D. L. Guris for discussion and critical reading of the manuscript.

This study was supported in part by grants from the National Institutes of Health (R01 DE015883) and the American Heart Association (0640109N) to A.I. and by the contracted research Protein 3000 Project by the Ministry of Education, Culture, Sports, Science and Technology of Japan to M.T. A.I. is an Established Investigator of the American Heart Association. J.S. is a Postdoctoral Fellow supported by the Greater Midwest Affiliate of the American Heart Association (0720073Z).

We declare that we have no competing financial interests.