title: Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphorylation.
abstract: The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (hbetac). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hbetac. We report here a comprehensive screen of the entire hbetac molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hbetac that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbetac mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbetac activation, dissociate hbetac tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbetac.
authors.affiliation: Hanson Centre for Cancer Research and Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia.
authors.forename: B J
authors.initials: BJ
authors.lastname: Jenkins
authors.forename: T J
authors.initials: TJ
authors.lastname: Blake
authors.forename: T J
authors.initials: TJ
authors.lastname: Gonda
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D053614:Janus Kinase 2; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008894:Milk Proteins; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D015245:Deoxyribonuclease BamHI; C024404:BglII endonuclease; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C018492:GCCNNNNNGGC-specific type II deoxyribonucleases; C029792:GTCGAC-specific type II deoxyribonucleases