title: Activation of Rac and tyrosine phosphorylation of cytokine receptors induced by cross-linking of integrin alpha4beta1 and cell adhesion in hematopoietic cells.
abstract: Adhesion of hematopoietic cells, mainly through alpha4beta1 and alpha5beta1 integrins, to the bone marrow microenvironment may play important roles in regulation of hematopoiesis. However, the mechanisms for signaling, outside-in signaling, have largely remained to be established. We demonstrate here that cross-linking of alpha4beta1 by anti-alpha4 antibody induces tyrosine phosphorylation of Pyk2, Shc, and Cbl as well as binding of the adaptor protein CrkL with Cbl in a murine hematopoietic cell line, 32D/EpoR-Wt. Furthermore, cross-linking of alpha4beta1 induced activation of the Rho family small GTPase Rac, which was enhanced by induced overexpression of CrkL and was inhibited by the phosphatidylinositol 3(')-kinase (PI3K) inhibitor LY294002. In addition, adhesion of 32D/EpoR-Wt cells to immobilized H-296, a recombinant fibronectin peptide specific for alpha4beta1, induced tyrosine phosphorylation of Jak2, the erythropoietin receptor (EpoR), and the IL-3 receptor beta subunit as well as Pyk2, Shc, and Cbl. Tyrosine phosphorylation of Jak2 and EpoR was also induced in a human leukemic cell line, UT-7, by adhesion to immobilized H-296. However, adhesion of 32D/EpoR-PM4 cells, expressing the W282R mutant EpoR defective in coupling with Jak2, to immobilized H-296 failed to induce tyrosine phosphorylation of the mutant EpoR. These results implicate CrkL in PI3K-dependent activation of Rac by outside-in signaling from alpha4beta1 and suggest that adhesion through alpha4beta1 further activates cytokine receptor-associated Jak2 to induce phosphorylation of these receptors.
authors.affiliation: Department of Hematology and Oncology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, 113-8519, Tokyo, Japan.
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Kanda
authors.forename: Zhen-Hua
authors.initials: ZH
authors.lastname: Jin
authors.forename: Daisuke
authors.initials: D
authors.lastname: Mizuchi
authors.forename: Ayako
authors.initials: A
authors.lastname: Arai
authors.forename: Osamu
authors.initials: O
authors.lastname: Miura
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D053614:Janus Kinase 2; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine / Q000737:chemistry / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D003432:Cross-Linking Reagents; D039041:Integrin alpha4beta1; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D017467:Receptors, Erythropoietin; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D020744:rac GTP-Binding Proteins