Molecular basis of crosstalk between oncogenic Ras and the master regulator of hematopoiesis GATA‐2

Abstract

Disease mutations provide unique opportunities to decipher protein and cell function. Mutations in the master regulator of hematopoiesis GATA-2 underlie an immunodeficiency associated with myelodysplastic syndrome and leukemia. We discovered that a GATA-2 disease mutant (T354M) defective in chromatin binding was hyperphosphorylated by p38 mitogen-activated protein kinase. p38 also induced multisite phosphorylation of wild-type GATA-2, which required a single phosphorylated residue (S192). Phosphorylation of GATA-2, but not T354M, stimulated target gene expression. While crosstalk between oncogenic Ras and GATA-2 has been implicated as an important axis in cancer biology, its mechanistic underpinnings are unclear. Oncogenic Ras enhanced S192-dependent GATA-2 phosphorylation, nuclear foci localization, and transcriptional activation. These studies define a mechanism that controls a key regulator of hematopoiesis and a dual mode of impairing GATA-2-dependent genetic networks: mutational disruption of chromatin occupancy yielding insufficient GATA-2, and oncogenic Ras-mediated amplification of GATA-2 activity.

GATA Factor-Dependent Positive-Feedback Circuit in Acute Myeloid Leukemia Cells

“…Ras(G12V) induced the slow mobility isoform of endogenous (Figure 1B) and expressed GATA-2 (Figures 1C and S1B). In our prior G1E proerythroblast and HEK293 analyses (Katsumura et al, 2014), Ras(G12V) stimulated GATA-2 S192 phosphorylation, inducing multi-site phosphorylation, which yields the slow-migrating isoform. S192A mutation abrogated Ras(G12V)-induced GATA-2 hyperphosphorylation in Kasumi-1 and Kasumi-3 cells (Figures 1C and S1B).…”

“…Previously, we described GATA-2 phosphorylation sites that create a slow mobility GATA-2 isoform detected by SDS-PAGE. We demonstrated that λ-phosphatase converts phosphorylated GATA-2 to a dephosphorylated, fast-migrating isoform (Katsumura et al, 2014). In Kasumi-1 cells, λ-phosphatase decreased the slow mobility phosphorylated isoform of endogenous GATA-2 (Figure 1A).…”

“…GATA2 mutations were detected in 7% of pediatric MDS patients (Wlodarski et al, 2016). These mutations attenuate GATA-2 chromatin binding, thus disrupting the GATA-2-dependent genetic network (Katsumura et al, 2014). Heterozygous mutations of a Gata2 intronic enhancer (+9.5 kb), which normally increases Gata2 expression in hemogenic endothelium, hematopoietic stem cells (HSCs), and myeloid progenitors (Gao et al, 2013; Grass et al, 2006; Johnson et al, 2012; Sanalkumar et al, 2014), cause MonoMAC with a phenotype resembling patients with coding region mutations (Hsu et al, 2013; Johnson et al, 2012).…”

“…Ras-p38 signaling stimulates GATA-2 S192 phosphorylation, which promotes multi-site GATA-2 phosphorylation and enhances GATA-2-mediated transcriptional activation in pro-erythroblast and endothelial cells (Katsumura et al, 2014). GATA-2 and oncogenic Ras cooperatively promote non-small-cell lung cancer and colon cancer (Kumar et al, 2012; Shen et al, 2014; Steckel et al, 2012).…”

Epigenetic Determinants of Erythropoiesis: Role of the Histone Methyltransferase SetD8 in Promoting Erythroid Cell Maturation and Survival

“…The microRNA 30 (miR-30) context Setd8 and Gata2 shRNAs were cloned into MSCV-PIG vector, kindly provided by Mitchell Weiss, using BglII and XhoI restriction sites. Retroviruses expressing shRNA targeting luciferase (control), SetD8, and Gata2, containing a Gata2 cDNA (56) or an empty vector, were produced by transfecting 3 ϫ 10 6 293T cells with 15 g of both MSCV-PIG vector and pCL-ECO viral packaging vector. Retrovirus-containing supernatant was harvested 24 or 48 h posttransfection.…”