Decreased E47 in Senescent B Cell Precursors Is Stage Specific and Regulated Posttranslationally by Protein Turnover

Abstract

The E2A-encoded transcription factor E47 is crucial to B lymphopoiesis. Senescent BALB/c mice (∼2 years old) had reduced pre-B cells ex vivo. Pro-B/early pre-B cells from these aged mice, both ex vivo and in vitro, were deficient in E47 protein. In vitro, IL-7 expanded pro-B/early pre-B cells from young BALB/c mice expressed E47 protein that was relatively stable over a 5-h period. Cultured senescent pro-B/early pre-B cells exhibited reduced E47 protein stability with ∼50–90% loss of E47 over the same time period. Degradation of E47 was effectively blocked by the proteasome inhibitor lactacystin as well as calpain I and II inhibitors; E2A proteins were also shown to undergo ubiquitination. Although senescent B cell precursors expressed less E47 protein, E47 mRNA levels and turnover were normal. Therefore, E47 protein levels are reduced relatively early in B lineage differentiation in senescence and the decline in E47 protein occurs via increased protein degradation by proteasome and, possibly, calpain pathways. In contrast, normal E47 protein levels were observed within the highly reduced pre-B cell pool in aged mice. This suggests that pre-B cells in senescence undergo selection based on E47 expression. Increased degradation rates and lower steady-state levels were also observed for the transcription factors Pax-5/BSAP, Bob-1, and Ikaros, but this was not a general property of all proteins in aged B cell precursors. Therefore, altered turnover of multiple, select proteins crucial to B cell development may contribute to diminished B lymphopoiesis in old age.

E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

“…Turnover of the E2A proteins is governed by the ubiquitin-proteasome pathway. Previous studies, mostly focused on E2A protein regulation and degradation during B-cell development, have suggested that ubiquitin-proteasome mediated E2A protein degradation is one of the important mechanisms involved in the regulation of their cellular abundance and biological activities (Nie et al, 2003;Van der Put et al, 2004;Riley et al, 2005). However, little information is available regarding the regulation and degradation of the E2A proteins during muscle differentiation.…”

Accelerated Notch-Dependent Degradation of E47 Proteins in Aged B Cell Precursors Is Associated with Increased ERK MAPK Activation

“…Densitometry values for desired proteins were normalized to that of the loading controls. In some experiments, normalized values of experimental proteins in old lysates (e.g., E47) were expressed relative to young controls (9,12).…”

“…Both transcription of the RAG enzymes and the surrogate L chain proteins in B cell precursors are, in part, mediated by the E2A-encoded transcription factors E47 and E12 (10). Previously, we have shown that E2A-encoded protein levels are reduced in B cell precursors from aged mice after their expansion with IL-7 in vitro (9,11,12). This was associated with reduction in the expression of 5 protein and reflected in decreased 5 and VpreB mRNAs (9).…”

“…This was associated with reduction in the expression of 5 protein and reflected in decreased 5 and VpreB mRNAs (9). The reduced steady-state levels of E47 proteins seen in cultured aged B cell precursors were demonstrated to result primarily from increased proteasome-mediated protein turnover (12).…”

“…We have previously shown that, in senescence, posttranscriptional mechanisms in B cell precursors generate reduced E47 protein expression (12); however, in activated splenic B cells reduced E47 mRNA stability is responsible for lower levels of E47 protein (13). In a recent report (14), the degradation of E47 proteins in lymphocytes was shown to be promoted by serine/threonine MAPKs and to be highly dependent upon activation of the Notch pathway.…”