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Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.

ABSTRACT: Production of mature erythrocytes requires multiple growth factors, but we do not know how their actions are coordinated. Here we show that erythroid progenitors from erythropoietin receptor (Epo-R)-/- fetal livers, infected in vitro with a retrovirus expressing the wild-type Epo-R, require addition of both Epo and stem cell factor (SCF) to form colony-forming unit erythroid (CFU-E) colonies. Thus, a functional interaction between KIT and the Epo-R, similar to what we reported in cultured cells, is essential for the function of CFU-E progenitors. In contrast, CFU-E colony formation in vitro by normal fetal liver progenitors requires only Epo; the essential interaction between activated KIT and the Epo-R must have occurred in vivo before or at the CFU-E progenitor stage. Using truncated dominant-negative mutant Epo-Rs, we show that KIT does not activate the Epo-R by inducing its dimerization, but presumably does so by phosphorylating tyrosine residue(s) in its cytosolic domain. By expressing mutant Epo-Rs containing only one of eight cytosolic tyrosines, we show that either tyrosine residue Y464 or Y479 suffices for Epo-dependent cell proliferation. However, only Epo-R F7Y479 is capable of supporting erythroid colony formation when expressed in (Epo-R)-/- fetal liver cells, indicating that Y464 either cannot send a differentiation signal or fails to respond to SCF/KIT activation. This work employs a novel experimental system to study the function of growth factors and their receptors in normal hematopoiesis.

SUBMITTER: Wu H

PROVIDER: S-EPMC19998 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.

Wu H H Klingmüller U U Acurio A A Hsiao J G JG Lodish H F HF

Proceedings of the National Academy of Sciences of the United States of America 19970301 5

Production of mature erythrocytes requires multiple growth factors, but we do not know how their actions are coordinated. Here we show that erythroid progenitors from erythropoietin receptor (Epo-R)-/- fetal livers, infected in vitro with a retrovirus expressing the wild-type Epo-R, require addition of both Epo and stem cell factor (SCF) to form colony-forming unit erythroid (CFU-E) colonies. Thus, a functional interaction between KIT and the Epo-R, similar to what we reported in cultured cells, ...[more]

PMID: 9050860

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Project description:AimTo investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells.MethodsReverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells.ResultsThe bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1, quinone oxidoreductase (NQO2), hydroxyisobutyrate dehydrogenase (HIBADH) and 14-3-3σ. For the subsequent coimmunoprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immunoprecipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc.ConclusionGenes of the proteins that interacted with 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.

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Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.

Publications

Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.

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