Sterol regulatory element-binding protein (SREBP)-1a is a transcription factor sensing cellular cholesterol levels and integrating gene regulatory signals mediated by MAP kinase cascades. Here we report the identification of serine 117 in SREBP-1a as the major phosphorylation site of the MAP kinases Erk1/2. This site was identified by nanoelectrospray mass spectrometry and peptide sequencing of recombinant fusion proteins phosphorylated by Erk1/2 in vitro. Serine 117 was verified as the major phosphorylation site by in vitro mutagenesis. Mutation of serine 117 to alanine abolished Erk2-mediated phosphorylation in vitro and the MAP kinase-related transcriptional activation of SREBP-1a by insulin and platelet-derived growth factor in vivo. Our data indicate that the MAP kinase-mediated effects on SREBP-1a-regulated target genes are linked to this phosphorylation site.Protein phosphorylation at serine and threonine residues is a key regulatory mechanism controlling proteins regulating metabolism, growth, differentiation, apoptosis, and gene expression of cells. One major class of serine/threonine kinases mediating signal transduction of various extracellular stimuli, including insulin and growth factors, are mitogen-activated protein kinases (MAPK) 1 (for review, see Ref. 1). Pathways involving MAPK consist of three kinases, i.e. MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK, which are sequentially activated. The activity of MAPK is stimulated by MKK-mediated dual phosphorylation (Thr-X-Tyr) in the activation loop. MKK is regulated by the serine/threonine kinase MKKK, which is linked by protein-protein interaction, phosphorylation, or subcellular relocalization to extracellular stimuli at the cell surface, similar to the receptor-associated tyrosine kinases of insulin and growth factors. Different MAPK cascades have been identified, but the best characterized in mammalian cells is the extracellular signal-regulated kinase (Erk) pathway leading to the activation of the MAPK isoforms Erk1 and Erk2. The majority of identified MAPK substrates are transcription factors regulating the expression of many genes (2). Transcription factors phosphorylated by activated Erk1/Erk2 are involved in hormone action (e.g. estrogen and glucocorticoid receptors), cell growth, and differentiation (e.g. Elk-1, Ets1, Sap-1a, c-Myc, STATs).Sterol-regulatory element binding proteins (called SREBP1a, SREBP-1c, and SREBP-2) are transcription factors that appear to transmit the signal of membrane-embedded cholesterol levels to the nucleus regulating the expression rate of multiple genes (3, 4). Recently, evidence is accumulating that SREBPs are not only involved in cholesterol-regulated events but are also gene regulatory targets of intracellular signaling pathways, e.g. MAP kinase cascades. In accordance with this hypothesis, we have previously shown that the effects of insulin and PDGF on LDL receptor promoter activity are abolished by a MAP kinase cascade inhibitor and are mediated via the SREBP-binding cis-element sre-1 (5, 6). Ov...

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The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo.

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