Department of Genetics, Cell Biology and Anatomy, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198.
University of Wisconsin-Madison Blood Research Program, Department of Cell and Regenerative Biology, Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53705.
J Biol Chem. 2020 May 15;295(20):7113-7125. doi: 10.1074/jbc.RA119.011895. Epub 2020 Apr 2.
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.
数百个无活性α基序（SAM）结构域具有预测的结构相似性，据报道可与蛋白质、脂质或 RNA 结合。然而，这些结构域中的大多数尚未进行功能分析。此前，我们证明含有 SAM 结构域的蛋白质 SAMD14 可促进 SCF/原癌基因 c-Kit（c-Kit）信号转导、红系祖细胞功能和红细胞再生。GATA2 和 SCL/TAL1 转录因子占据的 Samd14 增强子（Samd14-Enh）缺失会降低骨髓和脾脏中 SAMD14 的表达，并在溶血性贫血小鼠模型中导致致命性。为了严格确定 Samd14-Enh 缺失是否通过降低 SAMD14 水平来减少贫血依赖性 c-Kit 信号转导，我们在鼠 Samd14-Enh 原代红系前体细胞中开发了一种遗传挽救测定法。内源性水平的 SAMD14 表达可挽救 c-Kit 信号转导。保守的 SAM 结构域对于 SAMD14 增加集落形成活性、c-Kit 信号转导和祖细胞存活是必需的。为了阐明 SAM 结构域在 SAMD14 中的功能的分子决定因素，我们用预测结构相似的不同 SAM 结构域取代其 SAM 结构域。嵌合体在挽救信号转导、存活和集落形成活性方面的效果不如 SAMD14 本身。因此，SAMD14 的 SAM 结构域具有与其他 SAM 结构域不同的属性，并且是 SAMD14 作为细胞信号转导和红细胞再生调节剂的功能基础。
