title: Activation of phosphatidylinositol 3-kinase is necessary for differentiation of FDC-P1 cells following stimulation of type III receptor tyrosine kinases.
abstract: Signaling molecules that are responsible for proliferation and differentiation of hematopoietic cells following ectopic expression of receptor tyrosine kinases (RTKs) were investigated in the interleukin 3 (IL-3)-dependent hematopoietic cell line, FDC-P1. Cells were transfected with human platelet-derived growth factor receptor (PDGF-R), macrophage colony stimulating factor-1 receptor (CSF-1R), epidermal growth factor receptor (EGF-R), and chimeras consisting of the extracellular domain of EGF-R and the transmembrane and cytoplasmic domains of either HER2 (HER1-2) or c-kit (EK-R). All FDC-P1 transfectants proliferated in response to the corresponding growth factor in the absence of IL-3. However, only cells expressing PDGF-R, CSF-1R, and EK-R (type III RTKs) differentiated along the monocyte-macrophage lineage after treatment with their activating ligands. Analysis of proteins from these RTK-expressing cells revealed that a Mr 85,000 protein showed in vitro phosphorylation, and V8 protease peptide mapping showed that this protein was p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase). Accordingly, activation of PDGF-R-, CSF-1R-, and EK-R-expressing cells led to an increase in PI3-kinase activity. Expression of EK-R mutant Y721F, which lacked the known p85 binding site, blocked differentiation and activation of PI3-kinase, without affecting proliferation. Last, addition of wortmannin to cells expressing PDGF-R, CSF-1R, and EK-R blocked ligand-induced differentiation in a concentration-dependent manner, and this effect correlated with wortmannin's ability to inhibit PI3-kinase. Thus, ectopic expression of both type I and III RTKs could stimulate FDC-P1 proliferation in the absence of IL-3; however, only activation of type III RTKs led to differentiation via selective coupling to p85 and PI3-kinase activation.
authors.affiliation: Department of Molecular Biology, Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: Y
authors.initials: Y
authors.lastname: Kubota
authors.forename: T
authors.initials: T
authors.lastname: Angelotti
authors.forename: G
authors.initials: G
authors.lastname: Niederfellner
authors.forename: R
authors.initials: R
authors.lastname: Herbst
authors.forename: A
authors.initials: A
authors.lastname: Ullrich
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008024:Ligands; D010449:Peptide Mapping; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010748:Phosphopeptides / Q000032:analysis; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004791:Enzyme Inhibitors; D006133:Growth Substances; D007377:Interleukin-3; D008024:Ligands; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010748:Phosphopeptides; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; D000077191:Wortmannin