Identification and Characterization of EFhd2 Phosphorylation
Vázquez Rosa, Edwin F.
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EFhd2 is a calcium binding protein highly expressed in the central nervous system. Previous studies indicated that EFhd2 is associated to pathological forms of the microtubule- associated protein tau in JNPL3 tauopathy mouse model and Alzheimer’s disease (AD) patients. EFhd2 is a novel amyloid protein that is overexpressed and co-aggregates with hyperphosphorylated tau proteins in AD cases. However, the pathophysiological role of EFhd2 in tau-mediated neurodegeneration is unknown. Deregulated kinases that lead to the hyperphosphorylation of tau proteins is an important molecular event in tau-mediated neurodegeneration, since it facilitates the aggregation of tau proteins. Known tau kinases associated to neurodegeneration are Cdk5 and GSK3β. Based on the observation mentioned above, it was hypothesized that the neurodegeneration process can induce posttranslational modifications, like phosphorylation, on EFhd2. Our in vitro phosphorylation assay results demonstrate that Cdk5, but not GSK3β, phosphorylates EFhd2. Moreover, Cdk5 only phosphorylates EFhd2 one time. Tandem mass spectrometry analysis identified serine 74 (S74) as the phosphorylation site. A phospho-specific antibody against EFhd2 phosphorylated at S74 was generated and used to track the modification in AD postmortem brains. Results demonstrate a significant reduction in the amount of EFhd2 phosphorylated at S74 in AD patients compared with normal-aging patients. Next, we proceed to evaluate the effect of the phosphorylation on the association with Tau, formation of amyloid structures, and the calcium binding activity. No difference was observed in the association of EFhd2-tau when EFhd2 is phosphorylated at S74. However, a decrease in the calcium binding activity and a decrease in the formation of amyloid structures were observed when EFhd2 is phosphorylated at S74. The results suggest that the phosphorylation at S74 on EFhd2 is affected during the neurodegenerative process of AD. Further studies are necessary to identify the physiological and pathological roles of EFhd2 and the phosphorylation at S74.