Regulation of neurogenin stability by ubiquitin-mediated proteolysis

NGN (neurogenin), a proneural bHLH (basic helix–loop–helix) transcription factor, plays a central role in promoting neuronal specification and differentiation in many regions of the central nervous system. NGN activity has been shown extensively to be controlled at the transcriptional level. However, in addition, recent findings have indicated that the levels of NGN protein may also be regulated. In the present study, we have demonstrated that NGN protein stability was regulated in both Xenopus embryos and P19 embryonal carcinoma cells, a mammalian neuronal model system. In both systems, NGN was a highly unstable protein that was polyubiquitinated for destruction by the proteasome. NGN binds to DNA in complex with its heterodimeric E-protein partners E12 or E47. We observed that NGN was stabilized by the presence of E12/E47. Moreover, NGN was phosphorylated, and mutation of a single threonine residue substantially reduced E12-mediated stabilization of NGN. Thus E-protein partner binding and phosphorylation events act together to stabilize NGN, promoting its accumulation when it can be active. Abbreviations: (b)HLH, (basic) helix–loop–helix; cdk, cyclin-dependent kinase; CHX, cycloheximide; CKII, casein kinase II; GFP, green fluorescent protein; HA, haemagglutinin; HRP, horseradish peroxidase; IVT, in vitro translated; NeuroD, neurogenic differentiation; NGN, neurogenin; IVT, 35S-NGN, NGN IVT in the presence of 35S-methionine; NGNR1, NGN-related 1; Ni-NTA, Ni2+-nitrilotriacetate; NP40, Nonidet P40; UPS, ubiquitin–proteasome system; WT, wild-type