Biochemical and Phosphoproteomic Analysis of the Helix-Loop-Helix Protein E47

Abstract

Numerous in vitro as well as genetic studies have demonstrated that the activities of the E2A proteins are regulated at multiple levels, including modulation of DNA binding by the Id proteins, association with the transcriptional modulators p300 and ETO, and posttranslational modifications. Here, we use affinity purification of tagged E47 combined with mass spectrometry in order to show that E47 interacts with the entire ensemble of Id proteins, namely, Id1, Id2, Id3, and Id4. Furthermore, we find that the lysine-specific histone demethylase 1 (LSD1), the protein arginine N-methyltransferase 5 (PRMT5), the corepressor CoREST, and the chaperones of the 14-3-3 family associate with affinity-purified E47. We also identify a spectrum of amino acid residues in E47 that are phosphorylated, including an AKT substrate site. We did, however, find that mutation of the identified AKT substrate site by itself did not perturb B cell development. In sum, these studies show that the entire ensemble of Id proteins has the ability to interact with E47, identify factors that associate with E47, and reveal a spectrum of phosphorylated residues in E47, including an AKT substrate site.

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ACKNOWLEDGMENTS

We are grateful to Robert Rickert for the CD19^Cre and PTEN^flox/flox mice. We thank members of the Murre laboratory for reagents and stimulating discussions.

R.T. and L.Y.T.W. were supported by a training grant from the NIH (Cellular and Molecular Genetics). K.B was supported by the NIH. This work was supported by grants from the NIH to C.M.