Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta(1) integrin-mediated hematopoietic cell adhesion

The CrkL adaptor protein is involved in signaling from the receptor for ery thropoietin (Epo) as well as interleukin (IL)-3 and activates P, integrin-m ediated hematopoietic cell adhesion through its interaction with C3G, a gua nine nucleotide exchange factor for Rap1, We demonstrate here that Epo as w ell as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32 D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediat ed enhancement of cell adhesion was inhibited by expression of a dominant n egative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap 1A-63E, activated beta (1) integrin-dependent adhesion of hematopoietic cel ls. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetat e and ionomycin, which also enhanced cell adhesion to fibronectin, whereas U73122, an inhibitor of phospholipase C, inhibited both cytokine-induced ac tivation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as w ell as CrkL is involved in signaling from the EpoR endogenously expressed i n a human leukemic cell line, UT-7, These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as thro ugh additional pathways most likely involving phospholipase C gamma and str ongly implicate Rap1 in regulation of beta (1) integrin-mediated hematopoie tic cell adhesion.