Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion.
The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates beta(1) integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated beta(1) integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in 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 through additional pathways most likely involving phospholipase Cgamma and strongly implicate Rap1 in regulation of beta(1) integrin-mediated hematopoietic cell adhesion.
Arai
A
A
Department of Hematology and Oncology and the Department of Bioregulatory Medicine and Rheumatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, Tokyo 113, Japan.
Nosaka
Y
Y
Kanda
E
E
Yamamoto
K
K
Miyasaka
N
N
Miura
O
O
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors; D004921:Erythropoietin / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D005353:Fibronectins / Q000378:metabolism; D005799:Genes, Dominant; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D007377:Interleukin-3 / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051966:Phospholipase C gamma; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011760:Pyrrolidinones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D004791:Enzyme Inhibitors; D004963:Estrenes; D005353:Fibronectins; D020735:Guanine Nucleotide-Releasing Factor 2; D019012:Integrin beta1; D007377:Interleukin-3; D007476:Ionophores; D007527:Isoenzymes; D009687:Nuclear Proteins; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D011994:Recombinant Proteins; D004921:Erythropoietin; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D015759:Ionomycin; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D020827:rap1 GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate