Download Functional Characterization of the Interaction Between Alkali Cation/Proton Exchanger SLC9A6/NHE6 and Cyclin Dependent Kinase 5 (CDK5) Book in PDF, Epub and Kindle
"Loss-of-function mutations in the alkali cation/proton exchanger SLC9A6/NHE6 cause Christianson Syndrome (CS), a rare X-linked genetic disorder characterized by moderate to severe intellectual disability, epilepsy, ataxia, autistic behavior, and neurodegeneration. NHE6 is widely expressed but is especially abundant in nervous tissue where it localizes to vesicles along the recycling endosomal pathway of neurons and glia cells. Loss of NHE6 activity in cell and animal model systems results in excessive endosomal acidification, impaired cargo trafficking and signal transduction, as well as degeneration of certain neurons, though the molecular mechanisms underlying these phenomena are not fully resolved. We hypothesized that NHE6 mediates some of its effects through interactions of its C-terminal regulatory domain with cytoplasmic ancillary proteins, and that loss of these interactions is a contributing factor in the pathogenesis of CS. To this end, a yeast two-hybrid screen of a human brain cDNA library was performed by our group and lead to the identification of cyclin-dependent kinase 5 (CDK5) as a putative interacting partner of NHE6. CDK5 is a serine/threonine protein kinase expressed in most tissues, but primarily active in neurons. Loss of CDK5 expression results in a disease phenotype which closely resembles that observed for NHE6, including neuronal death, reduced neurite outgrowth, and increased susceptibility to seizures. In this study, we confirmed the formation of a NHE6 and CDK5 complex by biochemical (co-immunoprecipitation and glutathione S-transferase pull-down assays) and cell imaging (dual-labelling confocal microscopy and proximity ligation assay) analyses using Chinese hamster ovary AP-1 and neuroblastoma SH-SY5Y cells. CDK5 did not directly phosphorylate NHE6 in vitro despite the presence of a CDK5-like phosphor-acceptor site (S606PQA), though genetic manipulation of this site modestly affected CDK5 binding. Preliminary data from subcellular fractionation experiments suggest that NHE6 expression enhances CDK5 expression at the plasma membrane. However, the interaction between CDK5 and NHE6 is reduced in the presence of p35, an important activator of CDK5 that is tethered to the plasma membrane. Therefore, we propose that NHE6 serves as a scaffold for the endosomal delivery of CDK5 to the plasma membrane. There, CDK5 dissociates from NHE6 upon binding p35 and is now primed to activate neighboring effectors important for neuronal function"--