Lipid-Dependent Roles for SAMD14 in Stress Erythropoiesis

Sterile alpha motif-containing protein 14 (SAMD14) is upregulated in mouse models of anemia and its absence leads to lethality after severe hemolytic anemia, without affecting steady-state hematopoiesis. To elucidate how SAMD14 expression helps mice recover from acute stress, we conducted a global phosphoproteomic screen in Lin^-Kit⁺ cells from phenylhydrazine treated mice looking for post-translational modifications that are sensitive to SAMD14 levels. Phosphoproteins enriched in wildtype (WT) versus Samd14^ΔEnh/ΔEnh cells were involved in regulation of mTOR signaling, protein translation and cytoplasmic stress granule (SG) dynamics. SGs promote cell survival by sequestering mRNP complexes stalled in translation initiation, and SG formation relies on mTORC1-S6K1 activity. Interestingly, protein levels of phospho-EIF2α (Ser 51), which is tightly linked to SG formation, were 24-fold upregulated in anemia versus steady state. Our working hypothesis is that SAMD14 promotes survival of stress erythroid precursors (SEPs) by modulating stress granule dynamics. We tested by western blotting that proteins and phosphoproteins identified as Samd14-sensitive were also upregulated in acute anemia, including phospho-S6K1 (Thr 389) (3.9-fold), and SG-marker protein G3BP1 (2.4-fold), suggesting the involvement of SAMD14 in the mTOR pathway and SG formation.

We previously established a molecular interactor of SAMD14 (capping protein complex) which is a component of stress granules, but the interaction is not SAM-dependent. However, the SAMD14-SAM contains an evolutionarily conserved polybasic motif (PBM) predicted to bind phospholipids. Phospholipids are among the most enriched metabolites found in SGs. Additionally, phosphatidic acid and phosphatidyl 3-monophosphate (PI3P) have known roles in mTORC1 activation and phosphorylation of S6K1 which is important for SG formation. A PIP strip assay confirmed that Samd14-SAM binds PI3P. Deletion of SAM domain or disruption of PBM in S14-R381Q mutant reduced PI3P-binding affinity. Proximity ligation assays (PLA) showed decreased PLA signal in SAM-deleted mutant (3.3-fold) and S14R381Q (1.9-fold) compared to full length SAMD14. Further, we conducted colony assays to test the function of this interaction. Erythroid colonies (BFU-Es) are reduced 1.9-fold in erythroid precursor cells (EPCs) expressing the PBM-mutant compared to full-length. Thus, SAMD14-PI3P interaction promotes erythroid colony formation.

Consistent with the Samd14-dependent phosphoproteome results, PI3P regulates cellular processes like endocytic trafficking, autophagy, and mTOR signaling. We tested PI3P’s role in erythroid differentiation by inhibiting VPS34 activity, the sole phosphatidylinositol-3 kinase that synthesizes PI3P from phosphatidyl inositol. EPCs were differentiated to mature red blood cells in medium containing SAR405, a VPS34 inhibitor. Flow cytometry analysis with erythroid cell markers showed that VPS34 inhibition significantly increased the number of undifferentiated EPCs by 29% compared to DMSO controls. SAR405-treated cells express more cell surface KIT receptor (4-fold by flow cytometry), higher levels of Gata2 transcripts (1.5-fold by qPCR), and decreased globin levels (0.6-fold by qPCR), all characteristic of increased percentages of progenitor cells. This data suggests that VPS34 inhibition blocks terminal erythropoiesis which can be ascribed to PI3P’s prominent role in autophagy. Studies have shown that autophagy is crucial for SG clearance. Moreover, SEPs from PHZ-treated Samd14-knockout (KO) mice have 5.4-fold higher lipidated LC3B compared to WT as determined by immunoblotting. This suggests a defect in autophagic machinery in Samd14-KO SEPs possibly deregulating SG dynamics.

We discovered a completely novel mechanism in which SAMD14 upregulation in anemia promotes stress dependent mTOR activation, and potentially the frequency and function of stress granules. Ongoing experiments are testing whether the novel SAMD14-PI3P interaction controls mTORC1 activity and stress granule dynamics in mouse anemia models, using genetic complementation approaches with full length SAMD14 or S14R381Q expression constructs. This work will broaden our understanding of cellular pathways controlling SEP activation and survival after anemia and establish the mechanism by which SAMD14 alleviates anemia symptoms.