title: The multi-site docking protein Gab1 is constitutively phosphorylated independent from its recruitment to the plasma membrane in Jak2-V617F-positive cells and mediates proliferation of human erythroleukaemia cells.
abstract: The constitutively active Janus kinase 2 mutant Jak2-V617F is responsible for cytokine-independent growth of hematopoietic cells and the development of myeloproliferative neoplasms, such as polycythaemia vera and essential thrombocythaemia. Cells expressing Jak2-V617F exhibit constitutive STAT, MAPK, and PI3K signalling, and constitutive association of the multi-site docking protein Gab1 to PIP3 at the plasma membrane. Here, we demonstrate the crucial role of Gab1 for the proliferation of Jak2-V617F-positive human erythroleukaemia (HEL) cells. In Jak2-V617F-expressing cells Gab1 is constitutively phosphorylated by Erk1/2 on serine residue 552, which regulates binding to PIP3. Additionally, Gab1 is constitutively phosphorylated on tyrosine residue 627. Tyrosine 627 is a SHP2 binding site and required for Gab1-dependent Erk1/2 activation. As previously shown, Jak2-V617F-dependent Erk1/2 and PI3K activation act synergistically on the proliferation of Jak2-V617F-positive cells. Here, we examined whether constitutive membrane association of Gab1 explains cytokine-independent Gab1 phosphorylation in Jak2-V617F-expressing cells. Although we could demonstrate Jak2-V617F-dependent constitutive serine 552 and tyrosine 627 phosphorylation of Gab1, interestingly, both phosphorylations do not require binding of Gab1 to PIP3 at the plasma membrane. Instead, we observed a constitutive interaction of Gab1 with the erythropoietin receptor in Jak2-V617F-expressing cells, which depends on Janus kinase activity. Thus, constitutive Gab1-dependent signalling in Jak2-V617F-expressing cells does not occur due to the constitutive association of Gab1 with PIP3 at the plasma membrane.
authors.affiliation: Institute of Biology, Department of Systems Biology, Otto-von-Guericke University, Universitätsplatz 2, 39106 Magdeburg, Germany. Electronic address: hannes.bongartz@ovgu.de.
authors.forename: Hannes
authors.initials: H
authors.lastname: Bongartz
authors.affiliation: Institute of Biology, Department of Systems Biology, Otto-von-Guericke University, Universitätsplatz 2, 39106 Magdeburg, Germany. Electronic address: wiebke.hessenkemper@ovgu.de.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Hessenkemper
authors.affiliation: Department of Hematology and Oncology, Medical Center, Otto-von-Guericke University, Leipziger Straße 44, 39120 Magdeburg, Germany. Electronic address: christian.mueller@med.ovgu.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Müller
authors.affiliation: Institute of Biology, Department of Systems Biology, Otto-von-Guericke University, Universitätsplatz 2, 39106 Magdeburg, Germany. Electronic address: melissa.fensky@gmail.com.
authors.forename: Melissa
authors.initials: M
authors.lastname: Fensky
authors.affiliation: Institute of Biology, Department of Systems Biology, Otto-von-Guericke University, Universitätsplatz 2, 39106 Magdeburg, Germany. Electronic address: johannes.fritsch@boerde.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Fritsch
authors.affiliation: Institute of Molecular Medicine, Martin-Luther-University Halle-Wittenberg, ZAMED, Heinrich-Damerow-Straße 1, 06120 Halle, Germany. Electronic address: katharina.mandel@uk-halle.de.
authors.forename: Katharina
authors.initials: K
authors.lastname: Mandel
authors.affiliation: University of Luxembourg, Signal Transduction Laboratory, Life Sciences Research Unit, 6, avenue du Swing, L-4367 Belvaux, Luxembourg. Electronic address: iris.behrmann@uni.lu.
authors.forename: Iris
authors.initials: I
authors.lastname: Behrmann
authors.affiliation: University of Luxembourg, Signal Transduction Laboratory, Life Sciences Research Unit, 6, avenue du Swing, L-4367 Belvaux, Luxembourg. Electronic address: claude.haan@uni.lu.
authors.forename: Claude
authors.initials: C
authors.lastname: Haan
authors.affiliation: Department of Hematology and Oncology, Medical Center, Otto-von-Guericke University, Leipziger Straße 44, 39120 Magdeburg, Germany; Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke-University, Leipziger Straße 44, 39120 Magdeburg, Germany. Electronic address: thomas.fischer@med.ovgu.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Fischer
authors.affiliation: Institute of Molecular Medicine, Martin-Luther-University Halle-Wittenberg, ZAMED, Heinrich-Damerow-Straße 1, 06120 Halle, Germany. Electronic address: stephan.feller@uk-halle.de.
authors.forename: Stephan M
authors.initials: SM
authors.lastname: Feller
authors.affiliation: Institute of Biology, Department of Systems Biology, Otto-von-Guericke University, Universitätsplatz 2, 39106 Magdeburg, Germany. Electronic address: fred.schaper@ovgu.de.
authors.forename: Fred
authors.initials: F
authors.lastname: Schaper
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics* / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000473:pathology; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011087:Polycythemia Vera / Q000235:genetics* / Q000473:pathology; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D050791:STAT Transcription Factors / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013920:Thrombocythemia, Essential / Q000235:genetics* / Q000473:pathology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C098587:GAB1 protein, human; D018129:Phosphatidylinositol Phosphates; D017467:Receptors, Erythropoietin; D050791:STAT Transcription Factors; C060974:phosphatidylinositol 3,4,5-triphosphate; D019869:Phosphatidylinositol 3-Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2
keywords: Essential thrombocythaemia; Gab1; Jak2-V617F; Janus kinase; Myeloproliferative neoplasm; Polycythaemia vera