CrkL activates integrin-mediated hematopoietic cell adhesion through the guanine nucleotide exchange factor C3G.
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 (alpha4beta1) and VLA-5 (alpha5beta1) 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.
Arai
A
A
First Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
Nosaka
Y
Y
Kohsaka
H
H
Miyasaka
N
N
Miura
O
O
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D005353:Fibronectins; D005786:Gene Expression Regulation / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000502:physiology*; D051379:Mice; D009687:Nuclear Proteins / Q000502:physiology*; D011506:Proteins / Q000502:physiology*; D017469:Receptors, Fibronectin / Q000502:physiology*; D016201:Receptors, Lymphocyte Homing / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D020663:ras Guanine Nucleotide Exchange Factors; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D005353:Fibronectins; D020662:Guanine Nucleotide Exchange Factors; D039041:Integrin alpha4beta1; D016023:Integrins; D009687:Nuclear Proteins; D011506:Proteins; D017469:Receptors, Fibronectin; D016201:Receptors, Lymphocyte Homing; D020663:ras Guanine Nucleotide Exchange Factors