Characterization of the helix-loop-helix protein Id3

Members of the helix-loop-helix superfamily of proteins are involved in the regulation of cellular proliferation and differentiation during embryonic development. These proteins possess a conserved basic domain mediating DNA binding and a helix-loop-helix (HLH) domain mediating homo- or- hetero-dimerization between different HLH proteins. Id proteins are a subset of the HLH family which lack a basic region and are hypothesized to act as dominant negative regulators of DNA binding basic HLH proteins. All four Id family proteins share highly conserved amino acid sequences within the HLH domain but are divergent outside this region. In this study, we focused on the characterization of some of the biological activities of Id3 but in some instances we extended our analysis to the other members of the Id family.;First we demonstrate that Id proteins are capable of transcriptional activation when fused to the heterologous, GAL4 DNA binding domain. The HLH domain is necessary for the transactivation activity observed, suggesting that interaction with a cellular HLH protein is required. Cotransfection with exogenous Class A bHLH proteins (E proteins) greatly potentiates the transactivation, which is abolished upon cotransfection with Class B bHLH proteins. These results are consistent with the idea that the Id proteins have a transcriptional activity when present in a DNA binding complex.;We also show that the Id3 protein and the related Id1 and Id2 proteins are substrates for the ubiquitin-proteasome degradation pathway. In addition, we observed that coexpression of the bHLH protein E47 with Id3 significantly reduced the rate of degradation of Id3, suggesting that Id3 is less susceptible to degradation by the 26S proteasome when complexed to a bHLH protein.;Id proteins share some redundant functions, but we reasoned that each member of the family may also perform distinct functions mediated by interactions of the non-conserved regions of each protein with non-HLH proteins. By using the yeast two-hybrid system to identify Id3-interacting proteins, we identified the mouse homolog of human JAB1 protein. We characterized some of the properties of murine JAB1 (mJAB1). We show that the gene encoding the mJAB1 protein is localized on chromosome 1. mJAB1 mRNA is abundantly expressed in mouse embryos and adult tissues and JAB1 protein was readily detectable in many cell types.;We also demonstrate that Id3 and Id1 can interact with mJAB1 in mammalian cells. The Id3 interaction with mJAB1 is dependent upon the C-terminus of Id3, and does not require the HLH domain. We show that Id3, through its interaction with mJAB1, can suppress the ability of mJAB1 to potentiate the transactivation activity of c-Jun suggesting that the interaction of these two proteins in mammalian cells are functionally important. The results from these studies suggest that Id proteins may not always function in a dominant negative manner and that their diverse functions may reflect their presence in diverse protein complexes. Our data suggest that Id proteins are not only regulated differently but they may regulate different processes as revealed by their discrimination of interacting proteins. (Abstract shortened by UMI.)...