β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H

Abstract

CREB-H is an endoplasmic reticulum-tethered bZIP transcription factor, which critically regulates lipid homeostasis and gluconeogenesis in the liver. CREB-H is proteolytically activated by regulated intramembrane proteolysis to generate a C-terminally truncated form known as CREB-H-ΔTC, which translocates to the nucleus to activate the expression of target genes. We have previously characterized the roles and regulation of CREB-H in hepatic physiology and pathology. As the physiologically active form of CREB-H, CREB-H-ΔTC is a fast turnover protein. However, the mechanism governing CREB-H-ΔTC destruction was not well understood. In this study, I report on β-TrCP-dependent ubiquitination and proteasomal degradation of CREB-H-ΔTC. β-TrCP is the substrate recognition subunit of the E3 ubiquitin ligase SCF^(β-TrCP), which catalyzes the ubiquitination of various transcription factors and cell cycle regulatory proteins. By modulating the steady-state level and activity of these key factors, β-TrCP plays an important role in cell cycle control, oncogenesis and metabolic regulation. In my study, I first verified that the degradation of CREB-H-ΔTC was mediated by lysine 48-linked polyubiquitination. This degradation was effectively inhibited by a proteasome inhibitor. In search of the E3 ubiquitin ligase catalyzing CREB-H-ΔTC ubiquitination, bioinformatic analysis was carried out and β-TrCP was singled out as a potential E3. In light of the importance of β-TrCP in CREB-H-ΔTC degradation, experimental validation was performed. CREB-H-ΔTC physically interacted with β-TrCP. Forced expression of β-TrCP increased the polyubiquitination and decreased the stability of CREB-H-ΔTC, whereas knockdown of β-TrCP had the opposite effect. These results indicated that CREB-H-ΔTC undergoes β-TrCP-dependent lysine 48-linked polyubiquitination and degradation. I went on to characterize the molecular details of β-TrCP-mediated ubiquitination and degradation of CREB-H-ΔTC. An evolutionarily conserved sequence, SDSGIS, which functions as the β-TrCP-binding motif, was identified in CREB-H. CREB-H-ΔTC lacking this motif was stabilized and resistant to β-TrCP-induced polyubiquitination. This motif was a phosphodegron and phosphorylation at its serine residues was required for β-TrCP recognition. Furthermore, two additional phosphorylation sites close to the phosphodegron were identified. Interestingly, mutational analysis suggested that dephosphorylation of the two sites increased the transcriptional activity of CREB-H-ΔTC. Based on these results, I propose a new model for the regulation of CREB-H-ΔTC ubiquitination and degradation through both inhibitory and activating phosphorylation. Taken together, my work not only provided experimental evidence for β-TrCP-mediated ubiquitination and degradation of the active form of CREB-H transcription factor, but also revealed a new intracellular signaling pathway by which its ubiquitination and degradation are regulated. My findings have implications in the development of new agents that either stabilize or destabilize this important liver-enriched transcription factor.