CrkL Activates Integrin-Mediated Hematopoietic Cell Adhesion Through the Guanine Nucleotide Exchange Factor C3G

Abstract

CrkL is a member of the Crk family of adapter proteins consisting mostly of SH2 and SH3 domains. CrkL is most abundantly expressed in hematopoietic cells and has been implicated in pathogenesis of chronic myelogenous leukemia. However, its function has not been precisely defined. Here, we show that overexpression of CrkL enhances the adhesion of hematopoietic 32D cells to fibronectin. The CrkL-induced increase in cell adhesion was blocked by antibodies against VLA-4 (4β1) and VLA-5 (5β1) but was observed without changes in surface expression levels of these integrins. Studies using CrkL mutants demonstrated that the SH2 domain is partially required for enhancing cell adhesion, whereas the C-terminal SH3 domain as well as the tyrosine phosphorylation site (Y207) is dispensable. In contrast, the N-terminal SH3 domain, involved in binding C3G and other signaling molecules, was showed to play a crucial role, because a mutant defective of this domain showed an inhibitory effect on the cell adhesion to fibronectin. Furthermore, overexpression of C3G also increased the adhesion of hematopoietic cells to fibronectin, whereas a C3G mutant lacking the guanine nucleotide exchange domain abrogated the CrkL-induced increase in cell adhesion. On the other hand, a dominant negative mutant of H-Ras or that of Raf-1 enhanced the basal and CrkL-induced cell adhesion and that of R-Ras modestly decreased the adhesion. Taken together, these results indicate that the CrkL-C3G complex activates VLA-4 and VLA-5 in hematopoietic cells, possibly by activating the small GTP binding proteins, including R-Ras, through the guanine nucleotide exchange activity of C3G.

Gab1 mediates hepatocyte growth factor‐stimulated mitogenicity and morphogenesis in multipotent myeloid cells

“…Gab1 mediates signaling in the IL‐3, EPO, thrombopoietin and SCF pathways, all of which activate hematopoietic cell adhesion to FN through α 4 β 1 and α 5 β 1 integrins [Levesque et al, 1995]. Gab1 may promote this “inside‐out” integrin signaling by recruiting Crk‐L, which can activate integrin‐mediated 32D cell adhesion through the guanine nucleotide exchange factor C3G and the GTPase Rap1 [Arai et al, 1999; Sakkab et al, 2000]. Our observation of Gab1‐enhancement of IL‐3‐induced cell adhesion in hematopoietic cells is consistent with this model.…”

Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor

“…Integrin binding VLA-4/VLA-5/R-Ras [33,78,98] T-cell receptor c-Cbl/CrkL [99] B-cell receptor c-Cbl/CrkL [100] Insulin Cbl/Crk/C3G/TC10 [17,36,47] EGF EGF/Crk/C3G/Rap1/B-Raf [47,48] NGF FRS2/Crk/C3G /Rap1/B-Raf [14,48] Interferon γ c-Cbl/CrkL [101] Erythropoetin, interleukin-3 and interleukin-5 CrkL/STAT5 [102,103] Hepatocyte growth factor Gab1-CrkL [34] Growth hormone JAK2 and c-Src [18] Reelin stimulation via Dab1/CrkL [104] Mechanical force CrkII/Cas [60] Nectin c-Src/Crk [66] Cadherins c-Src/Vav2/Crk [105] Bombesin Crk/CrkL [106] TIMP2 Crk [81]…”

“…Expression of membrane-targeted C3G in HeLa cells also induces extensive cell spreading [77]. Overexpression of C3G in 32D cells increased adhesion to fibronectin through the activation of VLA-4 and VLA-5, mediated by R-Ras [78]. Overexpression of C3G increases adhesion of NIH 3T3 cells to laminin [79].…”

Papel de C3G plaquetario en angiogénesis y metástasis tumoral

“…Esto sugiere que su función no puede ser compensada por otros RapGEFs en estadios tempranos del desarrollo (Ohba et al, 2001). C3G participa a través de la activación de Rap1 en procesos de adhesión y migración mediados por integrinas (de Jong et al, 1998;Arai et al, 1999;Oh et al, 2004).…”