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 rec eptors, was cloned and found to be a physiologic regulator of platelet homeostasis. We report that megakaryocyte growth and development fact or (MGDF, thrombopoietin [TPO], c-mpl ligand) induces differentiation in a majority of mpl-transfected 32D cells, while interleukin (IL)-3 i s exclusively mitogenic in this system. MGDF differentiation, as measu red by decreased proliferation, changes in cellular morphology, increa sed adherence, and downregulation of very late antigen (VLA)-4, is dom inant over IL-3 proliferation. MGDF induces tyrosine-phosphorylation o f mpl, JAK2, SHC, SHPTP-1 (HCP, motheaten) and SHPTP-2 (Syp, PTP-1D) w ithin 30 seconds of stimulation, as well as of vav and MAPK with sligh tly delayed kinetics. A fraction of mpl and JAK2 is preassociated, and the stoichiometry of this complex is unaltered by cytokine stimulatio n. After MGDF stimulation, we detect interactions among SHC, grb2, SHP TP-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 J AK1 phosphorylation. Although similar in composition, the MGDF- and IL -3-induced complexes of signal transducers appear to be assembled in d ifferent configurations, especially with respect to SHPTP-2. Both MGDF and IL-3 induce tyrosine phosphorylation of STAT3 (APRF) and STATE (M GF), with MGDF favoring STAT3 while IL-3 predominantly causes STATE ph osphorylation. In addition, some proteins become tyrosine-phosphorylat ed in response to MGDF only, suggesting that we may have detected diff erentiation-specific signal transducers. These include a number of hig h-molecular-weight proteins (140 to 200 kD) and one 28-kD protein that becomes tyrosine-phosphorylated only briefly. (C) 1995 by The America n Society of Hematology.