Megakaryocyte growth and development factor and interleukin-3 induce patterns of protein-tyrosine phosphorylation that correlate with dominant differentiation over proliferation of mpl-transfected 32D cells.
Recently, the ligand for c-mpl, a member of the family of cytokine receptors, was cloned and found to be a physiologic regulator of platelet homeostasis. We report that megakaryocyte growth and development factor (MGDF, thrombopoietin [TPO], c-mpl ligand ) induces differentiation in a majority of mpl-transfected 32D cells, while interleukin (IL)-3 is exclusively mitogenic in this system. MGDF differentiation, as measured by decreased proliferation, changes in cellular morphology, increased adherence, and downregulation of very late antigen (VLA)-4, is dominant over IL-3 proliferation. MGDF induces tyrosine-phosphorylation of mpl, JAK2, SHC, SHPTP-1 (HCP, motheaten) and SHPTP-2 (Syp, PTP-1D) within 30 seconds of stimulation, as well as of vav and MAPK with slightly delayed kinetics. A fraction of mpl and JAK2 is preassociated, and the stoichiometry of this complex is unaltered by cytokine stimulation. After MGDF stimulation, we detect interactions among SHC, grb2, SHPTP-1, SHPTP-2, and the mpl/JAK2 complex. IL-3 induces phosphorylation of the above proteins with the exception of mpl and also causes weak JAK1 phosphorylation. Although similar in composition, the MGDF- and IL-3-induced complexes of signal transducers appear to be assembled in different configurations, especially with respect to SHPTP-2. Both MGDF and IL-3 induce tyrosine phosphorylation of STAT3 (APRF) and STAT5 (MGF), with MGDF favoring STAT3 while IL-3 predominantly causes STAT5 phosphorylation. In addition, some proteins become tyrosine-phosphorylated in response to MGDF only, suggesting that we may have detected differentiation-specific signal transducers. These include a number of high-molecular-weight proteins (140 to 200 kD) and one 28-kD protein that becomes tyrosine-phosphorylated only briefly.
Department of Developmental Biology, Amgen Inc, Thousand Oaks, CA 91320, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D007377:Interleukin-3 / Q000494:pharmacology*; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D016185:Receptors, Interleukin-3 / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013926:Thrombopoietin / Q000187:drug effects / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D007377:Interleukin-3; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D013926:Thrombopoietin; D011494:Protein Kinases