CrkL Mediates SDF-1-Induced Chemotaxis through Ras, Rac, and Erk Activation in Hematopoietic Cells.

The chemokine SDF-1 was originally identified as a growth promoting factor for pre-B cells and has been recognized to be indispensable for hematopoiesis from the findings on both its knock-out mice and those of its receptor CXCR4. SDF-1 is secreted by the bone marrow stromal cells and plays a crucial role for retention and homing of hematopoietic cells in the bone marrow through chemotaxis. In addition, SDF-1 regulates not only motility but also survival, proliferation, differentiation, and adhesion of the cells. Furthermore it was recently reported that leukemic cells show different responses to SDF-1 stimulation from normal hematopoietic cells. Despite its important roles in hematopoiesis, however, intracellular signaling pathways from CXCR4, especially those regulating chemotaxis of the hematopoietic cells, have largely remained to be clarified. We investigated the molecular mechanisms regulating chemotaxis induced by SDF-1 in hematopoietic cells and found that the enforced expression of adaptor molecule CrkL enhanced SDF-1-induced chemotaxis of murine hematopoietic cell lines, BAF3 and 32Dcl3, expressing CXCR4 on the cell surface. To examine the pathways up- and downstream of CrkL, we performed transient and stable transfection of CrkL and its mutants into BAF3 cell. Overexpression of CrkL enhanced SDF-1-induced Erk activation through Raf-1 and Mek. CrkL also enhanced SDF-1-induced activation of the Ras family GTPases Ras and Rap1 as well as the Rho family GTPase Rac. Among the dominant negative mutants of these small GTPases, those of Ras and Rac significantly suppressed CrkL-enhanced Erk activation. In addition, we also performed in vitro kinase assays using GST-CrkL as a substrate and found that SDF-1 induced phosphorylation of CrkL through the Src family tyrosine kinase Lyn. However, pretreatment with PP1, an inhibitor of Lyn, did not suppress activation of Ras, Rap1, Rac, and Erk at the concentration that suppressed the phosphorylation of CrkL. In addition, overexpression of a CrkL mutant that lacks the tyrosine targeted by Lyn did not reduce SDF-1-induced Erk activation. These results show that the phosphorylation of CrkL was not necessary for activation of the small GTPases and Erk. On the other hand, overexpression of a CrkL mutant lacking the N-terminal SH3 domain (CrkLdSH3N) required for the constitutive association with C3G, the guanine nucleotide exchange factor for Ras family GTPases, significantly reduced SDF-1-induced Erk activation. Therefore, this domain was required for the enhancement of SDF-1-induced Erk activation by CrkL through Ras. Finally we performed chemotaxis assays with transiently expressed mutants of these molecules. CrkLdSH3N as well as dominant negative forms of Ras and Rac reduced SDF-1-induced chemotaxis. Together, these results indicate that CrkL mediates SDF-1-induced chemotaxis of hematopoietic cells through activation of Ras, Rac, and the Raf/MEK/Erk pathway.