Phosphorylation Of Tau By Death-associated Protein Kinase1antagonizes The Kinase-induced Cell Apoptosis

Background:Alzheimer disease (AD) is an age-related neurodegenerative disorder characterized by the presence of senile plaques, neurofibrillary tangles and neuronal loss. The intracellular accumulation of the hyperphosphorylated tau plays an important role in neurodegeneration of AD, but the mechanism is not fully understood. Death-associated protein kinase1(DAPK1) is a member of the Ca2+/calmodulin-regulated family of serine/threonineprotein kinases. DAPK1has been shown to play a critical role as a kinase that can regulate cancer development, ischaemic-induced neuronal cell death and neurodegenerative disorders, and so on. Genome-wide association study and gene-centric single nucleotide polymorphisms have proved that DAPK1expression is closely related to late-onset Alzheimer’s disease. We have recently reported that phosphorylation of tau can antagonize cell apoptosis induced by staurosporine, camptothecin and hydrogen peroxide with the mechanisms involving preservation of β-catenin, however whether phosphorylation of tau can antagonize cell apoptosis induced by the intrinsically expressed factors in the brain DAPK1, is still not known.Aim:To explore the effects of DAPK1on tau phosphorylation and the role of tau phosphorylation in DAPK1-induced cell apoptosis.Methods:We detected the levels of DAPK1protein and tau phosphorylation in the brain of10month-old hTau mice and HEK293cells by immunohistochemistry or Western blotting. Wild type DAPK (wtDAPKl), siDAPKl, mutant DAPK1, siMARK2or its control vector was into HEK293cells with stable or transient expression of human tau441(HEK293/tau), DAPK1, tau phosphorylation, protein phosphatase2A (PP2A), glycogen synthase kinase-313(GSK-3B), protein kinase A (PKA), calcium/calmodulin dependent protein kinase Ⅱ (CaMKII), cdl division cycle2(Cdc2) and cyclin dependent protein kinase5(Cdk5) were detected by Western blotting; the cell viability was measured by using Cell Counting Kit-8(CCK-8). At last, the relationship between DAPK1and Tau protein was investigated by co-immunoprecipitationResults:We found that DAPK1protein was dramatically increased with a simultaneous hyperphosphorylation of tau at Thr231, Ser262, Ser396in hippocampal CA1, CA3, DG and the cortex of hTau mice, and the increased DAPK1staining were accumulated in the cytoplasmic compartment of the hTau mice. We observed that expression of tau could protect the serum deprivation-induced and DAPK1-mediated reduction of cell viability and caspase-3cleavage compared with the HEK293/wt cells, and phosphorylation of tau at Thr231, Ser262and Ser396was significantly increased. Manipulation of DAPKl did not change the levels of PP2A, GSK-3β, PKA, CaMKⅡ, Cdc2, Cdk5except phosphorylated MARK2. With downregulation of MARK2by shRNA, we did not see significantly change of tau phosphorylation at Thr231, Ser262and Ser396. We observed that DAPKl was associated with total tau proteins in HEK293/tau and in the hippocampal extracts of C57mice. Further studies by co-immunofluorescence labeling showed that DAPK1was co-localized with total tau and the Thr231-phosphorylated tau in HEK293/tau cells. Furthermore, expression of kinase domain-truncated DAPK1did not induce tau hyperphosphorylation at Thr231, Ser262and Ser396.Conclusion:Phosphorylation of tau protein at Thr231, Ser262and Ser396sites by death-associated protein kinase1antagonizes the kinase-induced cell apoptosis...