Fig. 4.

(A) Model depicting possible mechanism by which ESDN acts as a scaffold for the adaptor CrkL. ESDN’s ectodomains may respond to factors (X, Y, Z) that induce clustering of ESDN in the proximity of low activity SFKs. ESDN serves as a substrate for SFKs as well as unidentified tyrosine kinases at multiple tyrosine residues, some of which can bind to the SH2 domain of SFK’s and thereby relieve autoinhibition. Phosphotyrosine sites on ESDN serve as docking sites for CrkL and other phosphotyrosine-binding proteins. By virtue of primarily its N-terminal SH3 domain, CrkL may recruit additional effector proteins. (B–C) Antibody-induced clustering induces ESDN tyrosine phosphorylation and its interaction with the CrkL-SH2 domain. HEK 293 cells expressing wildtype or Y7F ESDN-Flag were treated as indicated with two μg/ml of an anti-ESDN antibody recognizing the extracellular Factor V/VIII domain. Cells were lysed and extracts were subjected to anti-Flag immunoprecipitations. Immune complexes and whole cells extracts were blotted with either anti-phosphotyrosine or anti-Flag antibodies as indicated. *Indicates non-specific band. (C) Cells were treated as in (B) except extracts were subjected to GST-CrkL-SH2 pulldowns prior to immunoblotting.