Phosphorylation of E47 as a Potential Determinant of B-Cell-Specific Activity

Abstract

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.

Ebf1-mediated down-regulation of Id2 and Id3 is essential for specification of the B cell lineage

“…The observation that Id2/Id3 mRNA levels increase in E47-expressing prepro-B cells suggest that some level of E47 must be transcriptionally competent and free from Id protein inhibition to transcriptionally activate these genes, although this level of ''free'' E2A is not sufficient to activate Ebf1 transcription. One possibility is that E47 may be posttranslationally modified by phosphorylation to stimulate homodimerization in a manner that is not inhibitable by Id proteins (25). Alternatively, E2A activation alone may not be sufficient to induce Ebf1 without cooperation from other regulatory factors stimulated by IL-7 receptor signaling.…”

“…In IL-7R␣Ϫ or IL-7-deficient mice, E2A proteins are expressed at wild-type levels and yet Ebf1 and Pax5 are absent, which suggests that other factors participate in Ebf1 induction in addition to E2A, that E2A requires posttranslational modification to activate Ebf1 expression, or that inhibitory proteins suppress the ability of E2A to activate Ebf1 expression in this context (23)(24)(25). The activation of Ebf1 seems to be the pivotal event in B cell fate specification in that Ebf1 can surprisingly rescue the B cell developmental program when overexpressed in the context of multiple gene knockout backgrounds that completely lack B-lineage cells including PU.1 (8), E2A (26), IL-7R␣ (23), and IL-7 (24).…”

Characterization of the mUBC9-binding Sites Required for E2A Protein Degradation

“…LexA-E2A(476 -494) and LexA-E2A(505-513) interacted strongly with TAD-mUbc9, yet there was no interaction with the intervening region, LexA-E2A(494 -504). Hybrid protein LexA-E2A(510 -520), which includes a casein kinase II phosphorylation site (14,15), and hybrid protein LexA-E2A(520 -532), which includes a cyclic AMP-dependent protein kinase site (15), showed no significant interaction with TAD-mUbc9. These results indicate that mUbc9 interacts with two distinct, nearby regions of the E2A proteins: E2A(476 -494) and E2A(505-513).…”

“…Consequently, E12 and E47 are regulated mainly by post-translational mechanisms. The Id family of HLH proteins sequester E12 and E47 into non-DNA-binding dimers (1, 2, 12, 13), and phosphorylation of E47 immediately upstream of the basic region inhibits DNA binding (14,15). Because the transmembrane receptor Notch inhibits full-length E47 by a mechanism independent of its bHLH and TADs, there may be additional E2A regulatory domains or cofactors (16).…”

Pearson Correlation Analysis of Microarray Data Allows for the Identification of Genetic Targets for Early B-cell Factor

“…5) (23). The lack of BCF1 is believed to be a result of lineagerestricted post-translational modifications of the E47 protein (43), so to achieve the formation of this protein complex in the progenitor cell, we utilized a forced-dimer of E47 (31) to generate stably transfected Ba/F3 cells expressing both BCF1 and EBF. One of these clones has been reported earlier (31), whereas three more (clones 3, 5 and 6, Fig.…”