Activation of Rac and tyrosine phosphorylation of cytokine receptors induced by cross-linking of integrin alpha4beta1 and cell adhesion in hematopoietic cells.
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.
Kanda
Eiichiro
E
Department of Hematology and Oncology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku, 113-8519, Tokyo, Japan.
Jin
Zhen-Hua
ZH
Mizuchi
Daisuke
D
Arai
Ayako
A
Miura
Osamu
O
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*
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