Lithium stabilizes the CCAAT/enhancer-binding protein alpha (C/EBPalpha) through a glycogen synthase kinase 3 (GSK3)-independent pathway involving direct inhibition of proteasomal activity.

CCAAT/enhancer-binding protein alpha (C/EBPalpha), a basic leucine zipper transcription factor, is involved in mitotic growth arrest and differentiation. Given that numerous proteins involved in cell cycle regulation are degraded via the ubiquitin-proteasome system, we examined whether the C/EBPalpha protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBPalpha is a short-lived protein with a half-life of approximately 1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBPalpha protein. Higher molecular weight species of ubiquitinated C/EBPalpha were detected in BALB/MK2, and in vitro studies confirmed that C/EBPalpha is degraded by the proteasome in an ATP- and ubiquitin-dependent manner. GSK3 is a known C/EBPalpha kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBPalpha protein levels, (ii) increased electrophoretic mobility of C/EBPalpha, and (iii) no increase in C/EBPalpha mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBPalpha may target it for proteasomal degradation. However, a mutant C/EBPalpha containing T to A mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl, and additional pharmacological inhibitors of GSK3 did not alter C/EBPalpha levels indicating the effects of LiCl on C/EBPalpha are GSK3-independent. LiCl treatment of BALB/MK2 cells inhibited C/EBPalpha degradation and produced a 6-fold increase in the half-life of C/EBPalpha protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBPalpha. These results demonstrate C/EBPalpha is degraded via a ubiquitin-dependent proteasomal pathway, and LiCl stabilizes C/EBPalpha through a GSK3-independent pathway involving direct inhibition of proteasome activity.