Role of Ca2+/Calmodulin dependent protein kinase IV on REST activity

The transcriptional repressor REST (Repressor Element 1-Silencing Transcription factor) is a zinc finger domain repressor protein which binds to the specific RE1 regulatory sequence in its target genes and limits their transcription by histone deacetylases and histone methylases recruitment. REST is involved in many key biological processes, including neural differentiation, neurogenesis, and titration of REST levels occurs during these processes. As key regulators of protein biology, post-translational modifications modulate protein structure, activity and localization. It has been demonstrated that REST protein degradation during neural differentiation could be regulated by ubiquitin-mediated proteolysis through a conserved phosphodegron, stressing the importance of the regulation of REST phosphorylation levels. This phosphorylation-mediated control of REST levels appears to be important also in pathologies. The principal aim of this study was to investigate whether REST expression may be modulated by calcium-signalling, a pivotal second messenger that regulates a variety of cellular processes, with special attention to calcium-dependent enzymes. By a computational bioinformatics analysis, we identified five phosphorylation sites for Ca2+/calmodulin-dependent kinases (CaMKs), a family of Serine/Threonine protein kinases that is responsible for mediation of many intracellular responses to elevated Ca2+. By pharmacological and genetic approaches, we demonstrated that CaM-Kinase IV (CaMKIV) exerts a negative post-translational regulation on REST protein stability in cortical neurons. We employed in our study a RESTGTinv-conditional animal model in which the expression of REST and its truncated forms are totally abolished upon Cre-recombinase activity. By specifically removing the expression of this transcription factor and/or interfering with kinase activities at the same time, we gain deeper details in REST functions and its possible regulation by CaMKIV. Our result shows that REST directly regulates synapse formation and activity, not impacting early stages of neurodevelopment. We also identified a role for both CaMKIV and REST in tuning the autophagic pathway, acting as negative and positive regulators respectively.Altogether, our data highlight an important interplay between CaMKIV and REST, which can be a crucial control mechanism regulating different aspects relevant in the physiopathology of neurons in a calcium-related manner.