Glycogen synthase kinase-3-mediated phosphorylation of serine 73 targets sterol response element binding protein-1c (SREBP-1c) for proteasomal degradation.

Qingming Dong,Rajendra Raghow,Marshall B Elam,Xiong Deng,Robert N Cole,Robert N O'Meally,Edwards A Park,Dave Bridges,Francesco Giorgianni,Sarka Beranova-Giorgianni

doi:10.1042/bsr20150234

Abstract

Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates genes involved in the de novo lipid synthesis and glycolysis pathways. The structure, turnover and transactivation potential of SREBP-1c are regulated by macronutrients and hormones via a cascade of signalling kinases. Using MS, we have identified serine 73 as a novel glycogen synthase kinase-3 (GSK-3) phosphorylation site in the rat SREBP-1c purified from McA-RH7777 hepatoma cells. Our site-specific mutagenesis strategy revealed that the turnover of SREBP-1c, containing wild type, phospho-null (serine to alanine) or phospho-mimetic (serine to aspartic acid) substitutions, was differentially regulated. We show that the S73D mutant of pSREBP-1c, that mimicked a state of constitutive phosphorylation, dissociated from the SREBP-1c-SCAP complex more readily and underwent GSK-3-dependent proteasomal degradation via SCF(Fbw7) ubiquitin ligase pathway. Pharmacologic inhibition of GSK-3 or knockdown of GSK-3 by siRNA prevented accelerated degradation of SREBP-1c. As demonstrated by MS, SREBP-1c was phosphorylated invitro by GSK-3β at serine 73. Phosphorylation of serine 73 also occurs in the intact liver. We propose that GSK-3-mediated phosphorylation of serine 73in the rat SREBP-1c and its concomitant destabilization represents a novel mechanism involved in the inhibition of de novo lipid synthesis in the liver.

Highlights

The sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates de novo lipogenesis in the liver by activating genes involved in fatty acid and triacylglycerol synthesis [1]
All SREBPs are synthesized as precursor proteins that are inserted into the endoplasmic reticulum (ER) where they associate with a chaperone, sterol-cleavage activating protein (SCAP) and ER retention proteins, insulin-induced gene (Insig)-1 and Insig-2 [3]
We have previously shown that insulin treatment led to a rapid phosphorylation of precursor SREBP (pSREBP)-1c and its ER to Golgi transport and regulated intra-membrane proteolysis (RIP) were tightly coupled to phosphorylation [25]

Summary

Introduction

The sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that regulates de novo lipogenesis in the liver by activating genes involved in fatty acid and triacylglycerol synthesis [1]. Cells were maintained in serum-free medium and extracts were prepared at 0, 2, 4, 6 and 8 h following cycloheximide treatment, size-fractionated by SDS-PAGE and subject to Western blot analysis with anti-HA antibodies to probe exogenously expressed pSREBP-1c and with anti-SREBP-1 antibodies to probe total pSREBP-1c- and nSREBP-1c-specific bands that were quantified by densitometry and Quantity One software.

Results

Conclusion