Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis

Wan Yee Leong,Hong Guo,Ou Ma,Hui Huang,Alan B Cantor,Alan D Friedman

doi:10.1074/jbc.m115.674234

Abstract

Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.

Highlights

Runx1/AML1 directs the emergence of long term adult hematopoietic stem cells from hemogenic endothelium during development and subsequently contributes to lymphoid, myeloid, and megakaryocyte lineage development [1,2,3,4]
We find that activated Src strongly increases Runx1 activity in 293T cells and that conversion of the five Runx1 tyrosines modified by Src to phenylalanine to generate Runx1(5F) markedly reduces activation of a model reporter in 293T cells, and Runx1(4F) induces Pu.1 or Cebpa less effectively than wild-type Runx1 in 32Dcl3 myeloid cells
Runx1 Tyrosine Phosphorylation Increases Trans-activation—The locations of the five Runx1 tyrosines modified by Src are diagrammed, and tyrosine clusters changed to aspartate (Asp), glutamate (Glu), or phenylalanine (Phe) are listed (Fig. 1A). (Runx1)4TKLUC, containing four Runx1 consensus sites positioned upstream of a minimal thymidine kinase promoter and luciferase cDNA, was co-transfected into 293T cells with CMV expression vectors for wild-type Runx1 (WT) or its 5F, 5D, 4F, 4D, 1F, or 1D variants (Fig. 1B, top)

Summary

Introduction

Runx1/AML1 directs the emergence of long term adult hematopoietic stem cells from hemogenic endothelium during development and subsequently contributes to lymphoid, myeloid, and megakaryocyte lineage development [1,2,3,4]. To determine whether the four C-terminal Runx1 tyrosines are phosphorylated in 32Dcl3 cells, protein extracts from two WT-ER or 4F-ER subclones with similar expression of total Runx1-ER proteins were subjected to IP with ER␣ antiserum followed by Western blotting for phosphotyrosine (Fig. 2C).

Results

Conclusion