Functional requirements for a Samd14-capping protein complex in stress erythropoiesis
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Evaluation Summary:
This is an interesting study describing the role of Samd14-capping protein (CP) complex in stress erythroid signaling. Using orthogonal cellular, biochemical and genetic complementation approaches, the authors provide evidence to establish a previously unrecognized mechanism for Samd14-CP interaction in regulating Kit signaling in erythroid regeneration in response to acute anemia. Findings of this work will be of broad interest to the study of erythropoiesis and cellular signaling.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals—involving cooperation between stem cell factor (SCF)/Kit signaling and other signaling inputs—are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the sterile alpha motif (SAM) domain 14 ( Samd14 ) gene increases the regenerative capacity of the erythroid system in a mouse genetic model and promotes stress-dependent Kit signaling. However, the mechanism underlying Samd14’s role in stress erythropoiesis is unknown. We identified a protein-protein interaction between Samd14 and the α- and β-heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP β subunit increased erythroid maturation in murine ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/Kit signaling in CD71 med spleen erythroid precursors. Given the roles of Kit signaling in hematopoiesis and Samd14 in Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.
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Evaluation Summary:
This is an interesting study describing the role of Samd14-capping protein (CP) complex in stress erythroid signaling. Using orthogonal cellular, biochemical and genetic complementation approaches, the authors provide evidence to establish a previously unrecognized mechanism for Samd14-CP interaction in regulating Kit signaling in erythroid regeneration in response to acute anemia. Findings of this work will be of broad interest to the study of erythropoiesis and cellular signaling.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Mechanisms that control erythroid precursors' response to stress signaling to replenish erythrocytes have important implications in acute/chronic anemia. Dr. Hewitt and his group recently described that Samd14 is required for the regenerative capacity of the erythroid system in response to acute hemolytic anemia. However, the interacting proteins and the mechanisms of Samd14 function remain largely known. In this manuscript, the authors first identified the actin capping proteins (CP) as Samd14-interacting complexes through an atypical CP binding (CPB) domain in erythroid cells. Using shRNA-mediated depletion of CP proteins, genetic complementation assays, and biochemical studies of protein interaction domains, the authors provide evidence that Samd14-CP interaction is a determinant of erythroid precursor …
Reviewer #1 (Public Review):
Mechanisms that control erythroid precursors' response to stress signaling to replenish erythrocytes have important implications in acute/chronic anemia. Dr. Hewitt and his group recently described that Samd14 is required for the regenerative capacity of the erythroid system in response to acute hemolytic anemia. However, the interacting proteins and the mechanisms of Samd14 function remain largely known. In this manuscript, the authors first identified the actin capping proteins (CP) as Samd14-interacting complexes through an atypical CP binding (CPB) domain in erythroid cells. Using shRNA-mediated depletion of CP proteins, genetic complementation assays, and biochemical studies of protein interaction domains, the authors provide evidence that Samd14-CP interaction is a determinant of erythroid precursor cell levels and functions by promoting SCF/c-Kit signaling. The authors further showed that the Samd14-CP interaction via CPB is not required for Epo signaling, indicating the stage- and signal-specific effects for Samd14-CP interaction in erythroid cell maturation.
Overall, this is a well-executed study describing a number of new findings related to the mechanistic and functional dissection of Samd14 in stress erythroid signaling. The detailed characterization of Samd14-CP interaction by IP-proteomics, genetic depletion experiments, and cellular and biochemical assays were well designed and executed, and the results support the main conclusions. A major strength of the current study is the rigorous dissection of the Samd14-CP interaction using orthogonal cellular, biochemical and genetic complementation approaches. This study establishes a previously unrecognized mechanism for Samd14-CP interaction and its functional role in regulating c-Kit signaling in the pathophysiology of erythroid regeneration in response to PHZ-induced acute hemolytic anemia. Thus, this work will be of broad interest to the study of erythropoiesis and cellular signaling.
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Reviewer #2 (Public Review):
In this manuscript, Ray and colleagues follow up on their interesting observation that SAMD14 is required for the response to acute anemia. Using mass spectrometry and co-IP, the authors demonstrate that SAMD14 interacts with components of the alpha capping complex (CP), and then they demonstrate that this interaction is not dependent on the SAM domain. They also use shRNA to study the role of the CP protein Capzb in terminal erythroid maturation. Based on the known roles of SAMD14 and the CP in signal transduction they then investigate the role of SAMD14-CP interactions in kit receptor and EPO signaling, using a combination of flow cytometry and colony forming assays. The manuscript depicts an interesting new pathway that may be important for augmenting KIT and EPO signaling in a maturation stage-specific …
Reviewer #2 (Public Review):
In this manuscript, Ray and colleagues follow up on their interesting observation that SAMD14 is required for the response to acute anemia. Using mass spectrometry and co-IP, the authors demonstrate that SAMD14 interacts with components of the alpha capping complex (CP), and then they demonstrate that this interaction is not dependent on the SAM domain. They also use shRNA to study the role of the CP protein Capzb in terminal erythroid maturation. Based on the known roles of SAMD14 and the CP in signal transduction they then investigate the role of SAMD14-CP interactions in kit receptor and EPO signaling, using a combination of flow cytometry and colony forming assays. The manuscript depicts an interesting new pathway that may be important for augmenting KIT and EPO signaling in a maturation stage-specific manner.
Strengths of this paper include using unbiased methods to identify SAMD14 interacting proteins, as well as the use of the genetic complementation to study the function of SAMD14 in stress erythroid progenitors.
The data in this paper is consistent with previous work suggesting an important role for SAMD14 in stress erythropoiesis, however some clarifications/extensions of the experiments are needed to more fully support the conclusions regarding the role of SAMD14-CP interactions in regulating erythroid maturation and augmenting kit and EPO signaling .
Additional data are needed to support the role of Capzb in erythropoiesis. The conclusion that shRNA knockdown of Capzb promotes erythroid maturation is based largely on CD71 levels (Fig 2B). As CD71 levels can be impacted by EPO signaling, cytospins would be helpful to support the role for Capzb in restraining erythroid maturation. Reporting on the viability and proliferation of the Capzb KD cells would also provide important insights into its role in erythroid maturation. A role for Capzb in the regulation of erythroid maturation could be further supported by overexpression studies.
Many of the conclusions in the manuscript are based on analyses of pERK or pAKT via flow cytometry in immunophenotypically defined populations of erythroblasts. While these assays are nicely done, functional assays of those specific immunophenotypic populations (colony forming ability, proliferation, etc) or alternate assessment of KIT/EPO signaling would help further support conclusions.
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Reviewer #3 (Public Review):
The manuscript from Ray et al. extends their previous findings that Samd14 acts downstream of the Kit receptor to augment signaling during stress erythropoiesis. The authors demonstrate that the interaction between the F actin capping (CP) complex and Samd14 is required for increased activation of Erk and AKT signaling pathways downstream of the Kit receptor.
Strengths. The strengths of this paper include the identification of an unknown component of the Kit receptor signaling pathway, the CP complex. The authors demonstrate this interaction and map the interaction domain on Samd14 using a careful and comprehensive set of experiments. The authors show convincingly that blocking this interaction affects Kit receptor signaling and the development of BFU-E and CFU-E in the spleen.
Weaknesses. The identification …
Reviewer #3 (Public Review):
The manuscript from Ray et al. extends their previous findings that Samd14 acts downstream of the Kit receptor to augment signaling during stress erythropoiesis. The authors demonstrate that the interaction between the F actin capping (CP) complex and Samd14 is required for increased activation of Erk and AKT signaling pathways downstream of the Kit receptor.
Strengths. The strengths of this paper include the identification of an unknown component of the Kit receptor signaling pathway, the CP complex. The authors demonstrate this interaction and map the interaction domain on Samd14 using a careful and comprehensive set of experiments. The authors show convincingly that blocking this interaction affects Kit receptor signaling and the development of BFU-E and CFU-E in the spleen.
Weaknesses. The identification of a new component of the Kit receptor signaling complex is an important result. However previous work showed that loss of Samd14 decreases Erk and Akt activity. This paper identifies the next step in this cascade but still shows us that Erk and Akt signaling are reduced. There are no data that address how Samd14 and the CP complex increase the activation of these pathways. The observation that knockdown of the CP complex leads to fewer BFU-E suggests that the role of the Samd14/CP complex may be earlier in stress erythropoiesis than the time points investigated. Further analysis of the role of this complex in stress erythropoiesis will need to be done.
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