ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation.

Induction of the human c-fos proto-oncogene by mitogens depends on the formation of a ternary complex by p62TCF with the serum response factor (SRF) and the serum response element (SRE). We demonstrate that Elk-1, a protein closely related to p62TCF in function, is a nuclear target of two members of the MAP kinase family, ERK1 and ERK2. Phosphorylation of Elk-1 increases the yield of ternary complex in vitro. At least five residues in the C-terminal domain of Elk-1 are phosphorylated upon growth factor stimulation of NIH3T3 cells. These residues are also phosphorylated by purified ERK1 in vitro, as determined by a combination of phosphopeptide sequencing and 2-D peptide mapping. Conversion of two of these phospho-acceptor sites to alanine impairs the formation of ternary complexes by the resulting Elk-1 proteins. Removal of these serine residues also drastically diminishes activation of the c-fos promoter in epidermal growth factor-treated cells. Analogous mutations at other sites impair activation to a lesser extent without affecting ternary complex formation in vitro. Our results indicate that phosphorylation regulates ternary complex formation by Elk-1, which is a prerequisite for the manifestation of its transactivation potential at the c-fos SRE.     

Elk-1 protein domains required for direct and SRF-assisted DNA-binding.

The Ets-related Elk-1 protein can bind to purine-rich DNA target sites in a sequence specific fashion and, in addition, can form a ternary complex with the c-fos serum response element (SRE) and the serum response factor (SRF). We demonstrate that Elk-1 can readily interchange between its different interaction partners. The amino terminal ETS-domain of Elk-1 was shown to be necessary and sufficient for direct DNA-binding activity. For ternary complex formation with the SRE and SRF, both the Elk-1 ETS-domain as well as flanking sequences up to amino acid 169 were required. Removal of sequences between the ETS-domain and amino acids 137-169 did not abolish ternary complex formation. This suggests the Elk-1 region spanning amino acids 137-169 to contain a protein-protein interaction domain. Furthermore, we have shown that a single amino acid exchange introduced into the ETS-domain can drastically alter the direct DNA-binding affinity of Elk-1 without severely affecting SRF-assisted binding to the SRE. Thus, Elk-1 requires different propensities of the ETS-domain to exert its different modes of DNA sequence recognition.     

Smooth muscle-specific genes are differentially sensitive to inhibition by Elk-1

Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases, such as atherosclerosis, restenosis, and asthma. In the current study, overexpression of Elk-1 in SMCs down-regulated expression of several endogenous smooth muscle-restricted proteins, including telokin, SM22α, and smooth muscle α-actin. In contrast, down-regulation of endogenous Elk-1 in smooth muscle cells increased the expression of only telokin and SM22α, suggesting that smooth muscle-specific promoters are differentially sensitive to the inhibitory effects of Elk-1. Consistent with this, overexpression of the DNA binding domain of Elk-1, which acts as a dominant-negative protein by displacing endogenous Elk-1, enhanced the expression of telokin and SM22α without affecting expression of smooth muscle α-actin. Elk-1 suppressed the activity of smooth muscle-restricted promoters, including the telokin promoter that does not contain a consensus Elk-1 binding site, through its ability to block myocardin-induced activation of the promoters. Gel mobility shift and chromatin immunoprecipitation assays revealed that Elk-1 binds to a nonconsensus binding site in the telokin promoter and Elk-1 binding is dependent on serum response factor (SRF) binding to a nearby CArG box. Although overexpression of the SRF-binding B-box domain of Elk-1 is sufficient to repress the myocardin activation of the telokin promoter, this repression is not as complete as that seen with an Elk-1 fragment that includes the DNA binding domain. In addition, reporter gene assays demonstrate that an intact Elk-1 binding site in the telokin promoter is required for Elk-1 to maximally inhibit promoter activity. Together, these data suggest that the differential sensitivity of smooth muscle-specific genes to inhibition by Elk-1 may play a role in the complex changes in smooth muscle-specific protein expression that are observed under pathological conditions.     

Regulation of miR-34 Family in Neuronal Development

Differentiation of neural stem cells (NSC’s) to mature and functional neurons requires coordinated expression of mRNA, microRNAs (miRNAs) and regulatory proteins. Our earlier unbiased miRNA profiling studies have identified miR-200, miR-34 and miR-221/222 as maximally up-regulated miRNA families in differentiating PC12 cells and demonstrated the capability of miR-200 family in inducing neuronal differentiation (J. Neurochem, 2015, 133, 640–652). In present study, we have investigated role of miR-34 family in neuronal differentiation and identified P53 as mediator of nerve growth factor (NGF) induced miR-34a expression in differentiating PC12 cells. Our studies have shown that NGF induced miR-34a, arrests proliferating PC12 cells to G1 phase, which is pre-requisite for neuronal differentiation. Our studies have also shown that increased expression of miR-34a controls the P53 level in differentiated PC12 cells in feedback inhibition manner, which probably prevents differentiated cells from P53 induced apoptosis. Expression profiling of miR-34 family in different neuronal, non-neuronal and developing cells have identified differentiated and aged brain cells as richest source of miR-34, which also indicates that higher expression of miR-34 family helps in maintaining the mature neurons in non-proliferative stage. In conclusion, our studies have shown that miR-34 is brain enriched miRNA family, which up-regulates with neuronal maturation and brain ageing and co-operative regulation of P53 and miR-34a helps in neuronal differentiation by arresting cells in G1 phase.