Association between Lyn protein tyrosine kinase (p53/56lyn) and the beta subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human megakaryocytic leukemia cell line (M-07e).
The role of the lyn product (p53/p56lyn), a membrane-associated protein tyrosine kinase in the signaling pathway used by granulocyte macrophage-CSFR (GM-CSFR) was investigated by using the GM-CSF-dependent human megakaryoblastic leukemia cell line M-07e. M-07e cells express GM-CSFR and are dependent on GM-CSF for survival and proliferation in vitro. Treatment with anti-lyn Abs coimmunoprecipitated, along with lyn product, the beta subunit of GM-CSFR and a phosphoprotein with a molecular mass of 120 kDa (p120) in the lysates of M-07e cells but not in the lysates of human monocyte-derived macrophages (HMDM) or human lymphoid leukemia cells. That the 120-kDa phosphoprotein coimmunoprecipitated by anti-lyn Abs is the beta subunit of GM-CSFR was confirmed in the immunoprecipitates (IP) of M-07e cells with the use of an agarose-conjugated anti-p-tyr mAb. The formation of GM-CSF/GM-CSFR/lyn signaling complexes was verified in an autoradiographic study with anti-lyn IP of M-07e cells that had been bound with 125I-labeled recombinant human (rh)GM-CSF. The p120 protein (beta subunit) was not detected in the IP of M-07e cells with anti-fyn or anti-PI3 Abs. A direct association of Lyn kinase with the beta subunit of GM-CSFR was illustrated with a reversed approach showing the recovery of Lyn protein in anti-beta (CRS1) but not anti-alpha IP of M-07e cells that had been starved for a prolonged period. Finally, the interaction of Lyn kinase with the GM-CSFR complexes was further corroborated using anti-GM-CSF (G133) mAb, which coimmunoprecipitated both the p120 beta subunit and lyn product in the lysates of M-07e cells that had been bound with rhGM-CSF before cell lysis. Removal of rhGM-CSF from culture medium for 10 to 12 h resulted in a marked decrease in lyn-associated kinase activity but not the beta subunit/lyn kinase complex formation. Taken together, our results showed that, in M-07e cells, Lyn protein tyrosine kinase (p53/p56lyn) is stably associated with a constitutively phosphorylated beta subunit of the GM-CSFR in a manner that seems to be independent of lyn kinase activity.
Li
Y
Y
Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Shen
B F
BF
Karanes
C
C
Sensenbrenner
L
L
Chen
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001345:Autoradiography; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D051381:Rats; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases
D000911:Antibodies, Monoclonal; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases