title: Proliferation signaling and activation of Shc, p21Ras, and Myc via tyrosine 764 of human granulocyte colony-stimulating factor receptor.
abstract: The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF-mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF-containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF-mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF-driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.
authors.affiliation: Institute of Hematology, Erasmus University and Dr. Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.
authors.forename: J P
authors.initials: JP
authors.lastname: de Koning
authors.forename: A A
authors.initials: AA
authors.lastname: Soede-Bobok
authors.forename: A M
authors.initials: AM
authors.lastname: Schelen
authors.forename: L
authors.initials: L
authors.lastname: Smith
authors.forename: D
authors.initials: D
authors.lastname: van Leeuwen
authors.forename: V
authors.initials: V
authors.lastname: Santini
authors.forename: B M
authors.initials: BM
authors.lastname: Burgering
authors.forename: J L
authors.initials: JL
authors.lastname: Bos
authors.forename: B
authors.initials: B
authors.lastname: Lowenberg
authors.forename: I P
authors.initials: IP
authors.lastname: Touw
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D016259:Genes, myc; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000166:cytology; D011506:Proteins / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000502:physiology*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000737:chemistry
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016179:Granulocyte Colony-Stimulating Factor; D014443:Tyrosine; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)