Regulation of Repressor Element-1 Silencing transcription factor (REST) by Calmodulin-dependent Kinase IV.

Repressor-element 1 silencing transcription factor (REST) is a zinc-finger-containing protein that has a vast array of target genes, including those involved in neuronal differentiation, neuronal activity, homeostatic plasticity, neuroinflammation and senescence. Understanding the endogenous mechanisms that control the level of REST is critical, if the protective expression of REST in neurodegeneration or aging were to become the focus of therapeutic approaches. Calcium is a signaling molecule that regulates neuronal activities as well as cellular processes such as autophagy leading to senescence and/or apoptosis in neurons. Therefore, calcium-sensitive activation of Calmodulin-dependent kinase family was investigated as putative kinases that may phosphorylate REST. Here we show results that CaMKIV, a nuclear kinase in its active form, regulates REST, which is subsequently degraded via a proteasome-independent manner. The putative CaMKIV phosphorylation sites are contained in the linker peptides of Cys His domains that reduces the affinity upon phosphorylation. Mass spectrometry analysis to identify the phosphopeptide(s) in myc-REST by recombinant CaMKIV is currently under investigation. In addition, we examine whether wild type and phosphomimic mutants of REST (T294E and S322E) show altered affinity to RE-1 sequence of mouse Nav1.2 gene by: 1) electromobility shift assays and 2) by molecular dynamics simulations of the 4 to the 8 zinc-finger domains of REST in complex with the RE-1 sequence. We propose that phosphorylation of REST by CaMKIV may homeostatically regulate the activity of REST according to increase in intracellular-calcium concentration for homeostatic response or for the maintenance of cell survival in neuronal cells.