title: Multiple phosphorylation sites are important for RUNX1 activity in early hematopoiesis and T-cell differentiation.
abstract: RUNX1 is essential for definitive hematopoiesis and T-cell differentiation. It has been shown that RUNX1 is phosphorylated at specific serine and threonine residues by several kinase families. However, it remains unclear whether RUNX1 phosphorylation is absolutely required for its biological functions. Here, we evaluated hematopoietic activities of RUNX1 mutants with serine (S)/threonine (T) to alanine (A), aspartic acid (D), or glutamic acid (E) mutations at phosphorylation sites using primary culture systems. Consistent with the results of knockin mice, RUNX1-2A, carrying two phospho-deficient mutations at S276 and S293, retained hematopoietic activity. RUNX1-4A, carrying four mutations at S276, S293, T300, and S303, showed impaired T-cell differentiation activity, but retained the ability to rescue the defective early hematopoiesis of Runx1-deficient cells. Notably, RUNX1-5A, carrying five mutations at S276, S293, T300, S303, and S462, completely lost its hematopoietic activity. In contrast, the phospho-mimic proteins RUNX1-4D/E and RUNX1-5D/E exhibited normal function. Our study identifies multiple phosphorylation sites that are indispensable for RUNX1 activity in hematopoiesis.
authors.affiliation: Department of Hematology & Oncology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Yoshimi
authors.forename: Susumu
authors.initials: S
authors.lastname: Goyama
authors.forename: Masahito
authors.initials: M
authors.lastname: Kawazu
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakagawa
authors.forename: Motoshi
authors.initials: M
authors.lastname: Ichikawa
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Imai
authors.forename: Keiki
authors.initials: K
authors.lastname: Kumano
authors.forename: Takashi
authors.initials: T
authors.lastname: Asai
authors.forename: James C
authors.initials: JC
authors.lastname: Mulloy
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Kraft
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Takahashi
authors.forename: Naoki
authors.initials: N
authors.lastname: Shirafuji
authors.forename: Mineo
authors.initials: M
authors.lastname: Kurokawa
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology*; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006410:Hematopoiesis / Q000235:genetics / Q000276:immunology*; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D010449:Peptide Mapping / Q000379:methods; D010766:Phosphorylation / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
chemical_list: D050676:Core Binding Factor Alpha 2 Subunit; C493726:Runx1 protein, mouse