Signal transduction of the human granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins.
Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells through their interaction with specific cell-surface receptors. There is a considerable degree of overlap in the biological effects of these two growth factors, but little is known about the mechanisms of postreceptor signal transduction. We have investigated the effects of GM-CSF and IL-3 on protein tyrosine-kinase activity in a human cell line, MO7E, which proliferates in response to either factor. Tyrosine-kinase activity was detected using immunoblotting with a monoclonal antibody (MoAb) specific for phosphotyrosine. GM-CSF and IL-3 were found to induce a nearly identical pattern of protein tyrosine phosphorylation using both one- and two-dimensional gel electrophoresis. Tyrosine phosphorylation of two cytosolic proteins in particular was increased more than 10-fold, a 93-Kd protein (pp93) and a 70-Kd protein (pp70). Tyrosine phosphorylation of pp93 and pp70 was observed within 1 minute, reached a maximum at 5 to 15 minutes, and gradually decreased thereafter. Other proteins of 150, 125, 63, 55, 42, and 36 Kd were also phosphorylated on tyrosine in response to both GM-CSF and IL-3, although to a lesser degree. Tyrosine phosphorylation was dependent on the concentration of GM-CSF over the range of 0.1 to 10 ng/mL and on IL-3 over the range of 1 to 30 ng/mL. Stimulation of MO7E cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) or cytokines such as G-CSF, M-CSF, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma, tumor necrosis factor (TNF), or transforming growth factor-beta (TGF-beta) did not induce tyrosine phosphorylation of pp93 or pp70, suggesting that these two phosphoproteins are specific for GM-CSF-or IL-3-induced activation. The extent and duration of phosphorylation of all the substrates were increased by pretreatment of cells with vanadate, an inhibitor of protein-tyrosine phosphatases. Importantly, culture of MO7E cells with vanadate (up to 10 mumol/L) resulted in a dose-dependent increase in GM-CSF-or IL-3-induced proliferation of up to 1.8-fold. These results suggest that tyrosine phosphorylation may be important for GM-CSF and IL-3 receptor-mediated signal transduction and that cell proliferation may be, at least partially, regulated by a balance between CSF-induced protein-tyrosine kinase activity and protein-tyrosine phosphatase activity.
Kanakura
Y
Y
Dana-Farber Cancer Institute, Boston, MA 02115.
Druker
B
B
Cannistra
S A
SA
Furukawa
Y
Y
Torimoto
Y
Y
Griffin
J D
JD
D000911:Antibodies, Monoclonal / Q000276:immunology; D001853:Bone Marrow / Q000473:pathology / Q000648:ultrastructure; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D007377:Interleukin-3 / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D007947:Leukemia, Megakaryoblastic, Acute / Q000097:blood / Q000201:enzymology / Q000473:pathology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000502:physiology*; D016184:Receptors, Colony-Stimulating Factor; D011971:Receptors, Immunologic / Q000378:metabolism / Q000502:physiology*; D016185:Receptors, Interleukin-3; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003115:Colony-Stimulating Factors; D006133:Growth Substances; D007377:Interleukin-3; D011956:Receptors, Cell Surface; D016184:Receptors, Colony-Stimulating Factor; D011971:Receptors, Immunologic; D016185:Receptors, Interleukin-3; D014638:Vanadates; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases