Mutation of serine 21 does not influence binding to DNA. Expression vectors encoding wild-type (WT) or S21A or S21D mutant C/EBPα were transiently transfected into COS-7 cells, and nuclear lysates were prepared. (A) Immunoblot analysis of wild-type C/EBPα and serine 21 mutant forms. (B) Gel shift analysis with nuclear lysates and a labeled oligonucleotide corresponding to the C/EBPα binding site from the human G-CSF receptor promoter. Supershifting was performed with antibody (ab) to C/EBPα.

RA-induced granulopoiesis in U937 cells is partially inhibited by C/EBPα-S21D. (A) U937 cells were stably transfected with expression vectors for C/EBPα-ER fusion proteins in which serine 21 of C/EBPα was replaced with alanine or aspartate. C/EBPα-ER-S21A (S21A) and C/EBPα-ER-S21D (S21D) expressing stable lines were left untreated or treated with 10 μM RA, 1 μM β-estradiol (Est), or 10 μM RA-1 μM β-estradiol (RA + Est). Two days later, cells were analyzed for NBT reduction. (B) Quantification of NBT staining from experiment described in panel A. (C) U937 cells expressing C/EBPα-ER-S21D were treated with RA at the indicated concentrations in the absence (−) or presence (+) of 1 μM β-estradiol. Two days later, RNA was collected and analyzed by Northern blotting with probes for the G-CSF receptor (GCSFR), C/EBPɛ, and GAPDH. (D) U937 cells expressing C/EBPα-ER-S21A were treated with RA at the indicated concentrations in the absence (−) or presence (+) of 1 μM β-estradiol (Est). Two days later, RNA was collected and analyzed by Northern blotting with probes for the G-CSF receptor, C/EBPɛ, and GAPDH.

C/EBPα-S21D is unable to induce granulopoiesis in K562 cells. (A) K562 cells were stably transfected with C/EBPα-ER fusion proteins encoding either wild-type (WT) C/EBPα or a C/EBPα mutant form in which serine 21 was replaced with alanine (S21A) or aspartate (S21D). Lysates from the parental K562 line (−) and two independent stable lines for each C/EBPα-ER fusion protein (wild-type and S21Aand S21D mutant forms, as indicated) were separated by SDS-PAGE and subjected to immunoblot analysis with an antibody against C/EBPα. (B) C/EBPα-ER-expressing stable lines from panel A were left untreated (control [Con]) or treated with 1 μM β-estradiol (Est) for 24 h. Cells were fixed and visualized by immunofluorescence with an antibody to C/EBPα. (C) C/EBPα-ER-expressing stable lines from panel A were left untreated (Con) or treated with 1 μM β-estradiol (Est) for 3 days, at which point cells were analyzed for NBT reduction. (D) Cells were scored for reduction of NBT, and quantified data are presented. (E) C/EBPα-ER-expressing stable lines from panel A were left untreated (−) or treated with 1 μM β-estradiol (+) for 3 days. RNA was collected and analyzed by Northern blotting with probes for the G-CSF receptor (GCSFR), C/EBPɛ, and GAPDH.

Phosphorylation of serine 21 alters the conformation of C/EBPα dimers. Expression vectors encoding wild-type (WT) or S21A or S21D mutant C/EBPα fused at the N or C terminus with CFP or YFP were constructed. Various pairwise combinations of C/EBPα-fluorophore fusion proteins were transfected into 3T3-L1 cells. Differences in FRET relative to wild-type C/EBPα in untreated cells are indicated by an asterisk (P < 0.05). (A) CFP at the C terminus and YFP at the N terminus of wild-type or S21A or S21D mutant C/EBPα. Con, control. (B) CFP and YFP at the N terminus of wild-type or S21A or S21D mutant C/EBPα. (C) CFP and YFP at the C terminus of wild-type or S21A or S21D mutant C/EBPα. Two days after transfection, cells were treated with 10 nM TPA or vehicle for 20 min and images were collected. Analysis of FRET between CFP and YFP is described in Materials and Methods. Effect of TPA on energy transfer between fluorophores (FRET slope) is represented graphically for each C/EBPα dimer combination. (D) Model illustrating that C/EBPα bends such that the N and C termini are in close proximity and that when phosphorylated at serine 21, the transactivation domain of C/EBPα moves away from the N and C termini of the other molecule in the dimer.

Phosphorylation of serine 21 in vivo. (A) CV-1 cells were transfected with an expression vector carrying the gene for either wild-type (WT) or S21A mutant C/EBPα. Lysates were separated by SDS-PAGE and subjected to immunoblot analysis with a phosphospecific antibody designed against serine 21 of C/EBPα (P-S21; top) or total C/EBPα (bottom). (B) After serum deprivation, 3T3-L1 adipocytes (Ads) were not treated (−) or treated (+) with insulin for 10 min. Lysates were separated by SDS-PAGE and subjected to immunoblot analysis for total C/EBPα (bottom) or C/EBPα phosphorylated on serine 21 (top) (C) Lysates from white adipose tissue (WAT), lung, bone marrow (BM), liver, and spleen were separated by SDS-PAGE and subjected to immunoblot analysis with antibody to phosphorylated serine 21 (P-S21) or C/EBPα. (D) U937 cells were washed twice in RPMI medium without serum and suspended in RPMI medium (no serum) at 10⁶/ml. After 2 h at 37°C, cells were treated with 10 μM U0126 (U0) or 50 μM PD98059 (PD) for an additional 2.5 h. TPA was added to a final concentration of 1 μM, and cells were incubated for an additional 30 min. Immunoblot analysis with antisera specific for phosphorylated serine 21 or for C/EBPα was then performed. For all experiments, similar results were obtained in three independent experiments.

Phosphorylation of C/EBPα on serine 21. (A) In vivo labeling of C/EBPα was performed to identify phosphorylated residues. ³²P-labeled C/EBPα was purified, cleaved with trypsin, and separated by high-performance liquid chromatography. Fraction 87 was found to contain radioactivity, and sequencing revealed it to be amino acids S16 to R35 of C/EBPα (x axis). The radioactivity associated with each amino acid was quantified (y axis; counts per minute [CPM]). The majority of the radioactivity was found in the sixth cycle, corresponding to serine 21. (B) Schematic representation of C/EBPα proteins engineered for further experiments. Four known phosphoamino acids, S21 (identified herein), T222, T226, and S230 (previously identified [37]) are illustrated. WT, wild type. (C) 3T3-L1 cells were infected with retroviruses carrying the genes for ΔC/EBPα and ΔC/EBPα-S21A. Cells were not treated (−) or treated (+) with 10% calf serum (CS) for 10 min prior to lysis and purification of nuclear proteins. Nuclear lysates were separated by SDS-PAGE and analyzed for C/EBPα by immunoblotting. (D) Samples from panel C were separated by isoelectric focusing (IEF) and analyzed for C/EBPα by immunoblotting. Acidic (+) and basic (−) ends are indicated. Similar results were obtained in at least three independent experiments.

ERK1/2 phosphorylates C/EBPα on serine 21. (A) 3T3-L1 cells were infected with a retrovirus carrying the gene for ΔC/EBPα. ΔC/EBPα-expressing cells were pretreated for 15 min in the absence (−) or presence (+) of the MEK inhibitor U0126 (10 μM) prior to treatment with vehicle (Con), 10% calf serum (CS), 25 ng of PDGF per ml, or 100 nM TPA for an additional 10 min. Lysates were separated by SDS-PAGE and subjected to immunoblot analysis for C/EBPα, ERK1/2, or phospho-ERK1/2. ERK1, which migrates with an apparent mobility of 44 kDa, is the upper doublet, and ERK2, which migrates with an apparent mobility of 42 kDa, is the lower. (B) Schematic representation of C/EBPα and a C/EBPα docking site mutant engineered for use in further experiments. The ERK docking site and serine 21 are shaded. The asterisk (*) indicates the phenylalanine that is converted to alanine in the docking site mutant form. WT, wild type. (C) 3T3-L1 cells were infected with retroviruses carrying the genes for wild-type and F31A mutant ΔC/EBPα. Cells were not treated (Con) or treated with 10% calf serum (CS) or 25 ng of PDGF per ml for 10 min. (D) In vitro kinase assays were performed with recombinant ERK2 and GST fusion proteins containing the first 139 amino acids of C/EBPα (WT) or the same region with the indicated point mutations (S21A and F31A). Proteins separated by SDS-PAGE were stained with Coomassie blue (bottom) and subjected to autoradiography (top).