P-1a did not intefere with protein/DNA interaction Phosphorylation of SREBP-1a by JNK and p38 Kinases 3 Phosphorylation of SREBP-1a by JNK and p38 Kinases 4 Phosphorylation of SREBP-1a by JNK and p38 Kinases SREBP-1a-NT and in vitro kinase assays to monitor the effect of phosphorylation on protein/DNA interaction. We incubated in vitro transcribed and translated His-SREBP-1a-NT without or with JNK1 or p38a and subsequently analyzed the protein/DNA interaction by EMSA. SREBP-1a-NT interacted with sre-1 as well as E-box 
motif to a similar degree. Phosphorylation of SREBP-1a-NT affected protein/DNA interaction neither by JNK1, nor by p38a. This is in accordance to the observation that ERK phosphorylation of SREBP-1a at S117 does not influence protein/DNA interaction. JNK and p38 MAPK target the identified phosphorylation sites of SREBP-1a in intact cells To test the relevance of identified phoshorylation sites for JNK as well as p38 MAPK more specificily in cellular context we performed promoter reporter gene assays recruiting the heterologous Gal4-system and specific kinases for the MAP kinases cascades. In this assay, SREBP-1a-NT and the mutated forms were expressed as fusion proteins with the DNA binding domain of yeast Gal4. The latter domain alone doesn’t have any trans-activity per se. Accordingly, activities detected reflect trans-activities of SREBP-1a-NT or its mutants fused to the yeast Gal4 domain. To activate JNK and p38 signalling selectively in Hep G2 cells, the AT 7867 constitutively active upstream activators for JNK, i.e. MKK4DE, or p38, i.e. MKK3DE, were used. To monitor the specific initiation of JNK and p38 signaling to SREBP-1a in HepG2 cells MKK4DE or MKK3DE were cotransfected with wildtype pFA SREBP 1a NT or respective phosphorylation site mutants. Basal trans-activities of all mutated forms of SREBP-1a-NT were comparable to wildtype. Constitutive active MKK4DE or MKK3DE synergistically elevated transactivity of SREBP-1a-NT by about 3-fold. Transfection of activated MKK3 or MKK4 showed that S117A specifically interferes with MKK4 signaling, whereas S63A and T426V abolished MKK3 signaling. Accordingly the triple mutant failed to be activated by both kinase pathways. These results clearly show that SREBP-1a was a specific target for JNK and p38 activation, since the specific upstream kinases trigger kinase signaling to the specificphosphorylation sites. Relevance of SREBP-1a phosphorylation in vivo To investigate the biological relevance of MAPK related phosphorylation of human SREBP 1a in vivo, we generated transgenic mice which express selectively in liver the mutant variant of human SREBP-1a lacking all identified major phosphorylation sites, designated as SREBP-1aDP. In addition we generated transgenic mice which express liver specifically the wild type PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189542 of human SREBP-1a to a similar degree. Our intent was to study the role of SREBP 1a phosphorylation in liver for the phenotype of mice. Taken into consideration the complex and highly regulated releasing cascade of mature SREBP-1a protein from ER anchoured precursor proteins and to circumvent secondary cholesterol feedback effects we used the Nterminal transcriptional active domain. To facilitate detection a HA-tag was added and for liver specific expression the albumin promotor and a liver specific enhancer was chosen. The HA-tag did not affect phosphorylation, trans-activation or protein/DNA interaction of the Nterminal transcriptional active domain of SREBP-1a in vitro.