The Samd14 Sterile Alpha Motif Domain Promotes Stress-Induced Cellular Signaling and Survival

More than 200 mammalian proteins contain Sterile Alpha Motif (SAM) domains. While some of these domains are reported to mediate protein, lipid or RNA binding, the majority have not been analyzed. Our prior work discovered that Samd14, a SAM-domain containing protein, was transcriptionally activated by the GATA2 and Scl/TAL1-occupied Samd14 enhancer (Samd14-Enh). Deletion of Samd14-Enh lowers Samd14 expression in mouse bone marrow and spleen and causes lethality in a mouse model of severe hemolytic anemia. In anemia, stress erythroid progenitors respond to a multitude of paracrine signals, including erythropoietin (Epo) and stem cell factor (SCF), to induce rapid expansion and differentiation until homeostasis is re-established. Mechanistic analyses revealed that Samd14 regulates SCF/c-Kit signaling, erythroid progenitor function and promotes erythrocyte regeneration in anemia. Ex vivo, Samd14-Enh^-/- erythroid progenitors (CD71⁺Ter119^-Kit⁺) exhibited 2.1-fold and 1.6-fold lower phospho (Serine 473) AKT (pAKT) vs. WT in response to 5 min and 10 min SCF stimulation, respectively. To rigorously establish whether the Samd14-Enh deletion reduces anemia-dependent c-Kit signaling by lowering Samd14 levels in erythroid progenitors, we restored Samd14 expression in Samd14-Enh^-/- primary erythroid precursor cells. Defective SCF/c-Kit signaling in Samd14-Enh^-/- spleen progenitors could be rescued by reestablishing expression of Samd14. To test the role of the SAM domain in Samd14-mediated promotion of stress-induced erythroid progenitor function, we generated a SAM-domain deleted construct of Samd14 (Samd14 Δ SAM) to replace endogenous expression in cells from Samd14-Enh-deleted bone marrow and spleen ex vivo. In colony assays, full-length Samd14 increased GFP⁺ colony formation 2.7-fold, whereas there was no significant increase in colonies when expressing Samd14 Δ SAM vs. EV. Compared to expression of full-length Samd14, Samd14 Δ SAM exhibited 1.9-fold fewer (p=0.0006) BFU-E colonies (Figure 4B). Together, these results indicated that the Samd14 SAM domain is required for maximal promotion of colony forming ability, cell signaling and survival of erythroid progenitors. As the Samd14 SAM domain mediates SCF/c-Kit signaling, and cells lacking Samd14-Enh have impaired c-Kit signaling following anemia, this protein motif controls anemia-dependent erythroid progenitor cell genesis and/or function. Ongoing analyses to fuse the SAM structural domains of related proteins Neurabin-1 and SHIP-2 will test the sequence requirements of the Samd14 SAM domain on c-Kit signaling and stress erythroid progenitor function. These findings reveal a vital SAM domain-dependent molecular mechanism in stress erythroid progenitors whereby a GATA2 and anemia-activated protein facilitates SCF/c-Kit signaling during regenerative erythropoiesis. Given the importance of GATA2 and GATA2-dependent mechanisms in hematopoiesis, determining the role of the GATA2-Samd14-c-Kit axis in hematologic diseases may reveal unique functions.