Generation of Truncated C/EBPβ Isoforms by in VitroProteolysis

Abstract

Multiple forms of the transcriptional regulator CCAAT/enhancer-binding protein ␤ (C/EBP␤) with molecular masses of approximately 38, 34, 20, and 14 kDa have been observed in cell extracts. It has been proposed that these proteins arise by alternative initiation at in-frame AUG codons. The truncated C/EBP␤ isoforms (p14 and p20/LIP) lack transactivation domains but retain DNA-binding and dimerization sequences and are therefore assumed to function as competitive inhibitors of C/EBP-mediated transcription in vivo. By comparing various extraction procedures to analyze endogenous and overexpressed C/EBP␤ proteins, we determined that p20-C/EBP␤ is generated predominantly by in vitro proteolytic cleavage during isolation from cells and that p14-C/EBP␤ is produced exclusively by this mechanism. In transfected cells, the full-length (p34 and p38) isoforms but not the truncated proteins were detectable in the cytoplasm, indicating that the latter are not primary translation products. In addition, the C/EBP␤ leucine zipper dimerization domain was essential for the appearance of the truncated species, demonstrating that protein folding or dimerization are critical determinants of proteolytic sensitivity. Our findings suggest that the presence of truncated C/EBP␤ proteins in cell extracts must be interpreted with caution and that assumptions about the in vivo relevance of these isoforms should be re-evaluated.

MKP-1 antagonizes C/EBP β activity and lowers the apoptotic threshold after ischemic injury

“…When transfected in HN33 lines, WT C/EBPb generated all three major forms, including several smaller products generated through calpainmediated cleavage (p14, p20 and p20*). 18 In contrast, the DA and DC point mutants were defective in their expression of LAP1 and LIP, respectively ( Figure 7a). As seen before, transfection of GFP-MKP-1 induced nuclear pyknosis in HN33 cells cultured under normoxic conditions (10.0 ± 3.2% versus 40.5 ± 1.3%, Po0.001).…”

CCAAT/enhancer binding protein beta (C/EBPβ)-2 transforms normal mammary epithelial cells and induces epithelial to mesenchymal transition in culture

“…In previous studies, we did not observe elevated C/EBPb-3 (LIP) expression in any of the human breast tumor samples examined, although seven of the 10 tumors examined were high-grade, invasive mammary carcinomas (Eaton et al 2001). Since C/EBPb-3 is easily generated by artifactual proteolysis during extract preparation, it is possible that this may be the source of the discrepancy (Baer and Johnson, 2000). We did observe acquisition of C/EBPb-2 expression in the invasive mammary carcinoma samples we examined, which is noteworthy, given the ability of C/EBPb-2 expression to confer an invasive phenotype on MCF10A cells in culture.…”

“…Expression in normal tissue has only recently been documented (Eaton et al, 2001). As is the case with the smallest C/EBPb isoform, C/EBPb-3 or LIP, mechanisms involving both alternative translation initiation and proteolytic clipping may occur depending on the cell type and circumstances involved (Calkhoven et al, 2000;Xiong et al, 2001;Baer and Johnson, 2000). However, given the potential role that C/EBPb-2 may play in the genesis of metastatic breast cancer, it would be very valuable to begin to address the mechanism(s) of selective expression in normal and transformed mammary epithelial cells.…”

“…Thus, alternative translation of the C/EBPb-2 specific expression vector cannot occur (Calkhoven et al, 2000 andXiong et al, 2001). However, to demonstrate that expression of tagged-C/EBPb-2 in transduced cells did not result in elevated C/EBPb-3 levels via proteolytic cleavage (Baer and Johnson, 2000), Western analysis of whole cell lysates (WCLs) from untreated, LZRS-LacZ, or LZRS-His-C/EBPb-2-infected MCF10A cultures was performed (Figure 2d). The arrows in Figure 2d indicate the locations of the exogenous C/EBPb-2 bands detected in lane 3 containing LZRS-His-C/EBPb-2-infected MCF10A WCL.…”

“…The mammary gland phenotype of C/EBPb-null mice is more extensive than that observed in cyclin D1 knockout animals: the mammary epithelial cells (MECs) fail to proliferate in response to hormonal signals at puberty and during pregnancy, and the MECs fail to differentiate in response to lactationspecific hormones. Owing to alternative translation initiation at three in-frame methionines, the intronless C/EBPb gene (also termed NF-IL6 or CEBPB in humans and LAP or Cebpb in rodents) can generate three different protein products (Descombes and Schibler, 1991;Timchenko et al, 1999;Baer and Johnson, 2000;Calkhoven et al, 2000;and Xiong et al, 2001). The larger isoforms (termed LAP* and LAP in rodents) are transactivators.…”