Lithium Stabilizes the CCAAT/Enhancer-binding Protein (C/EBP ) through a Glycogen Synthase Kinase 3 (GSK3)-independent Pathway Involving Direct Inhibition of Proteasomal Activity*

CCAAT/enhancer-binding protein (C/EBP ), 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/EBP protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBP is a short-lived protein with a half-life of 1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBP protein. Higher molecular weight species of ubiquitinated C/EBP were detected in BALB/MK2, and in vitro studies confirmed that C/EBP is degraded by the proteasome in an ATPand ubiquitin-dependent manner. GSK3 is a known C/EBP kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBP protein levels, (ii) increased electrophoretic mobility of C/EBP , and (iii) no increase in C/EBP mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBP may target it for proteasomal degradation. However, a mutant C/EBP 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/EBP levels indicating the effects of LiCl on C/EBP are GSK3-independent. LiCl treatment of BALB/ MK2 cells inhibited C/EBP degradation and produced a 6-fold increase in the half-life of C/EBP protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBP . These results demonstrate C/EBP is degraded via a ubiquitindependent proteasomal pathway, and LiCl stabilizes C/EBP through a GSK3-independent pathway involving direct inhibition of proteasome activity.