Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Animals
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COS Cells
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Cell Adhesion
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Dose-Response Relationship, Drug
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Enzyme Inhibitors
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Erythropoietin / metabolism*
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Estrenes / pharmacology
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Fibronectins / metabolism
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Genes, Dominant
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Guanine Nucleotide-Releasing Factor 2 / metabolism
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Hematopoietic Stem Cells / cytology*
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Hematopoietic Stem Cells / metabolism
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Humans
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Integrin beta1 / metabolism*
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Interleukin-3 / metabolism*
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Ionomycin / pharmacology
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Ionophores / pharmacology
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Isoenzymes / metabolism
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Mice
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Mutation
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Nuclear Proteins / metabolism
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Phosphodiesterase Inhibitors / pharmacology
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Phospholipase C gamma
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Plasmids / metabolism
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Precipitin Tests
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Pyrrolidinones / pharmacology
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Recombinant Proteins / metabolism
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Signal Transduction
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Tetradecanoylphorbol Acetate / pharmacology
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Time Factors
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Transfection
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Tumor Cells, Cultured
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Type C Phospholipases / antagonists & inhibitors
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Type C Phospholipases / metabolism
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rap1 GTP-Binding Proteins / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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CRKL protein
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Enzyme Inhibitors
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Estrenes
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Fibronectins
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Guanine Nucleotide-Releasing Factor 2
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Integrin beta1
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Interleukin-3
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Ionophores
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Isoenzymes
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Nuclear Proteins
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Phosphodiesterase Inhibitors
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Pyrrolidinones
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Recombinant Proteins
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Erythropoietin
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1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
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Ionomycin
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Type C Phospholipases
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Phospholipase C gamma
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rap1 GTP-Binding Proteins
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Tetradecanoylphorbol Acetate