Sterile α-motif domain requirement for cellular signaling and survival

Abstract

Hundreds of sterile α-motif (SAM) domains have predicted structural similarities and are reported to bind proteins, lipids, or RNAs. However, the majority of these domains have not been analyzed functionally. Previously, we demonstrated that a SAM domain-containing protein, SAMD14, promotes SCF/proto-oncogene c-Kit (c-Kit) signaling, erythroid progenitor function, and erythrocyte regeneration. Deletion of a Samd14 enhancer (Samd14–Enh), occupied by GATA2 and SCL/TAL1 transcription factors, reduces SAMD14 expression in bone marrow and spleen and is lethal in a hemolytic anemia mouse model. To rigorously establish whether Samd14–Enh deletion reduces anemia-dependent c-Kit signaling by lowering SAMD14 levels, we developed a genetic rescue assay in murine Samd14–Enh−/− primary erythroid precursor cells. SAMD14 expression at endogenous levels rescued c-Kit signaling. The conserved SAM domain was required for SAMD14 to increase colony-forming activity, c-Kit signaling, and progenitor survival. To elucidate the molecular determinants of SAM domain function in SAMD14, we substituted its SAM domain with distinct SAM domains predicted to be structurally similar. The chimeras were less effective than SAMD14 itself in rescuing signaling, survival, and colony-forming activities. Thus, the SAMD14 SAM domain has attributes that are distinct from other SAM domains and underlie SAMD14 function as a regulator of cellular signaling and erythrocyte regeneration.

Exploring a diverse world of effector domains and amyloid signaling motifs in fungal NLR proteins

“…The clustering-based search revealed also one new annotation (assigned to 24 sequences), the Sterile Alpha Motif family SAM_Ste50p [55]. SAM motifs are involved in homologous and heterologous protein-protein interactions [56].…”

Functional Requirements for a Samd14-Capping Protein Complex in Stress Erythropoiesis

“…Given the role of Samd14 in stress-activated SCF/c-Kit signaling (Hewitt et al, 2017, Ray et al, 2020, we tested whether the Samd14 CPB mediates SCF-dependent activation of cell signaling in stress erythroid cultures. 48 hours after infection with either control (EV) or HA-Samd14 retrovirus, lineage-depleted splenocytes from PHZ-treated Enh -/mice were serum-starved, stimulated with SCF, and analyzed by flow cytometry for AKT and ERK activation.…”

“…Hemagglutinin (HA)-tagged full-length Samd14, Samd14 lacking the SAM domain (Samd14 ΔSAM) (Ray et al, 2020) Chinese Hamster Ovary cell conditioned medium. For retroviral infections, 1x10 6 lineagecells were added to 100 μl viral supernatant, polybrene (8 μg/ml) and HEPES buffer (10 μl/ml), and spinoculated at 2600 rpm for 90 min at 30°C.…”

“…To test whether CP function extends to adult stress erythropoiesis, akin to Samd14, we knocked down Capzb in the context of acute anemia induced by phenylhydrazine. Stress erythroid progenitors and precursors were isolated from WT spleen, cultured ex vivo, and monitored for erythroid differentiation, erythroid colony forming potential and survival (Figure 3A) (Ray et al, 2020). In contrast to Samd14 mRNA, which increases upon PHZ-induced anemia, Capzb and Capza2 mRNA decreased after PHZ treatment in a population containing erythroid precursors (defined by surface markers CD71 + Kit + Ter119 -) (Figure 3B).…”

“…SAMD14 promoter hypermethylation and/or downregulation is associated with adenocarcinoma (Sun, Iijima et al, 2008), gastric cancer (Xu, Chang et al, 2020) and the prostate tumor microenvironment (Teng, Pereira et al, 2021). In an ex vivo genetic complementation assay to restore Samd14 expression in Samd14-Enh -/precursors, an evolutionarily conserved SAM domain in SAMD14 functions to increase c-Kit signal transduction and erythrocyte regeneration (Ray, Chee et al, 2020). Despite structural similarities, the molecular features of the SAMD14 SAM domain are distinct from SAM domains in other proteins.…”