American Society of Hematology

Figure 1

$Figure 1. The relationship of autonomous homodimerization and signal activation to cell-survival capacity in Asn505 and ΔAsn505 mutants of c-Mpl. (A) Detection of homodimers and phosphorylation of intracellular signals in the Asn505 mutant of c-MPL. The “mock” group comprised Ba/F3 cells with the PCI-Neo vector, and acted as the negative control. Ser505 and Asn505 indicate Ba/F3 cells expressing wild-type (Ser505) or Asn505, respectively. The cross-linking assay was conducted under nonreducing conditions to detect dimers resulting from disulfide or hydrogen bonds. Noncleavable, water-soluble BS3 was used as a cross-linker to detect the homodimers. (The reaction product is an amine-containing compound with a spacer length of 11.4 Å.) The c-MPL monomeric (∼ 85 kDa) and dimerized bands (∼ 170 kDa) are indicated by ◀. The dimerized band was detected in Asn505-expressing Ba/F3 cells in the absence of TPO (top panel). The experiments were repeated using reducing conditions (with 5% 2-mercaptoethanol in the sample buffer) and gave the same results as under nonreducing conditions (data not shown). The phosphorylation status of MEK1/2 and STAT5 is shown in the middle panel. Phosphorylation of MEK1/2 and STAT5 was observed independently of TPO stimulation in Asn505-expressing cells, although the phosphorylated signals in TPO-free conditions were weaker than those in the presence of TPO. The bottom panel shows the expressions of the c-MPL in the membrane fractions, which were separated using Mem-PER Eukaryotic membrane protein extraction reagent kit (Thermo Scientific) in immunoblotting. The c-MPL expressions in the whole cell lysates, the membrane fractions, and the cytoplasmic fractions were examined, respectively, and only the gel images of the membrane fractions were picked up and aligned here. (B) Detection of homodimers and phosphorylation of intracellular signals in the truncated c-MPL. The ΔSer505-1 and ΔSer505-2 mutants have truncation of the entire ECD of the wild-type (Ser505) of c-MPL, while the ΔAsn505-1 and ΔAsn505-2 mutants have truncation of the entire ECD of the Asn505 mutants of c-MPL. The anti–c-MPL antibody (Upstate Biotechnology), used here for detection of dimerization, recognizes the C-terminal of c-MPL. Noncleavable membrane-permeable DSG was used as a cross-linker to detect homodimers of the ECD-truncated c-MPL mutants. (The reaction product is an amine-containing compound with a spacer length of 7.7 Å.) The cross-linking assay was conducted in the absence of factor (IL-3 or TPO) stimulation. The truncated c-MPL monomers (∼ 20 kDa) and dimers (∼ 40 kDa) are indicated by ◀. Dimerized bands were detected in both ΔAsn505-1 and ΔAsn505-2, but not in either of the ΔSer505 mutants under nonreducing conditions (top panel). Vertical lines have been inserted to indicate a repositioned gel lane. MEK1/2 and STAT5 showed phosphorylation in both ΔAsn505 clones even in the absence of IL-3 stimulation (middle panel). The bottom panel shows the expressions of the truncated c-MPL in the membrane fractions in immunoblotting (see legend for panel A). The truncated c-MPL expressions in the whole cell lysates, the membrane fractions, and the cytoplasmic fractions were examined, respectively, and only the gel images of the membrane fractions were picked up and aligned here. (C) MTT assay of the c-MPL mutants without factor stimulation. Briefly, transfected Ba/F3 cells were washed in PBS and resuspended in RPMI medium containing 10% FBS. A total of 5 × 104 cells per well in 100 μL medium were plated in 96-well plates. A total of 10 μL thiazolyl blue tetrazolium bromide (5 mg/mL; Sigma-Aldrich) was used for daily estimation of the relative cell viability (%) over a period of 4 days. The results are shown as means ± SEM of 3 separate experiments. Asn505-1 and Asn505-2 are Ba/F3 transfectants expressing Asn505. (D) Schematic diagram of Ser505, Asn505, ΔSer505, and ΔAsn505 mutants of c-MPL, and the results of phosphorylation of intracellular signals. Two different cross-linkers, BS3 and DSG, were used here to detect homodimer formation; the lengths of the spacer are indicated (↔). A circled “P” indicates phosphorylation of intracellular signals (MEK1/2 and STAT5) with homodimer formation.$

The relationship of autonomous homodimerization and signal activation to cell-survival capacity in Asn505 and ΔAsn505 mutants of c-Mpl. (A) Detection of homodimers and phosphorylation of intracellular signals in the Asn505 mutant of c-MPL. The “mock” group comprised Ba/F3 cells with the PCI-Neo vector, and acted as the negative control. Ser505 and Asn505 indicate Ba/F3 cells expressing wild-type (Ser505) or Asn505, respectively. The cross-linking assay was conducted under nonreducing conditions to detect dimers resulting from disulfide or hydrogen bonds. Noncleavable, water-soluble BS³ was used as a cross-linker to detect the homodimers. (The reaction product is an amine-containing compound with a spacer length of 11.4 Å.) The c-MPL monomeric (∼ 85 kDa) and dimerized bands (∼ 170 kDa) are indicated by ◀. The dimerized band was detected in Asn505-expressing Ba/F3 cells in the absence of TPO (top panel). The experiments were repeated using reducing conditions (with 5% 2-mercaptoethanol in the sample buffer) and gave the same results as under nonreducing conditions (data not shown). The phosphorylation status of MEK1/2 and STAT5 is shown in the middle panel. Phosphorylation of MEK1/2 and STAT5 was observed independently of TPO stimulation in Asn505-expressing cells, although the phosphorylated signals in TPO-free conditions were weaker than those in the presence of TPO. The bottom panel shows the expressions of the c-MPL in the membrane fractions, which were separated using Mem-PER Eukaryotic membrane protein extraction reagent kit (Thermo Scientific) in immunoblotting. The c-MPL expressions in the whole cell lysates, the membrane fractions, and the cytoplasmic fractions were examined, respectively, and only the gel images of the membrane fractions were picked up and aligned here. (B) Detection of homodimers and phosphorylation of intracellular signals in the truncated c-MPL. The ΔSer505-1 and ΔSer505-2 mutants have truncation of the entire ECD of the wild-type (Ser505) of c-MPL, while the ΔAsn505-1 and ΔAsn505-2 mutants have truncation of the entire ECD of the Asn505 mutants of c-MPL. The anti–c-MPL antibody (Upstate Biotechnology), used here for detection of dimerization, recognizes the C-terminal of c-MPL. Noncleavable membrane-permeable DSG was used as a cross-linker to detect homodimers of the ECD-truncated c-MPL mutants. (The reaction product is an amine-containing compound with a spacer length of 7.7 Å.) The cross-linking assay was conducted in the absence of factor (IL-3 or TPO) stimulation. The truncated c-MPL monomers (∼ 20 kDa) and dimers (∼ 40 kDa) are indicated by ◀. Dimerized bands were detected in both ΔAsn505-1 and ΔAsn505-2, but not in either of the ΔSer505 mutants under nonreducing conditions (top panel). Vertical lines have been inserted to indicate a repositioned gel lane. MEK1/2 and STAT5 showed phosphorylation in both ΔAsn505 clones even in the absence of IL-3 stimulation (middle panel). The bottom panel shows the expressions of the truncated c-MPL in the membrane fractions in immunoblotting (see legend for panel A). The truncated c-MPL expressions in the whole cell lysates, the membrane fractions, and the cytoplasmic fractions were examined, respectively, and only the gel images of the membrane fractions were picked up and aligned here. (C) MTT assay of the c-MPL mutants without factor stimulation. Briefly, transfected Ba/F3 cells were washed in PBS and resuspended in RPMI medium containing 10% FBS. A total of 5 × 10⁴ cells per well in 100 μL medium were plated in 96-well plates. A total of 10 μL thiazolyl blue tetrazolium bromide (5 mg/mL; Sigma-Aldrich) was used for daily estimation of the relative cell viability (%) over a period of 4 days. The results are shown as means ± SEM of 3 separate experiments. Asn505-1 and Asn505-2 are Ba/F3 transfectants expressing Asn505. (D) Schematic diagram of Ser505, Asn505, ΔSer505, and ΔAsn505 mutants of c-MPL, and the results of phosphorylation of intracellular signals. Two different cross-linkers, BS³ and DSG, were used here to detect homodimer formation; the lengths of the spacer are indicated (↔). A circled “P” indicates phosphorylation of intracellular signals (MEK1/2 and STAT5) with homodimer formation.

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