A DNA damage-activated checkpoint kinase phosphorylates tau and enhances tau-induced neurodegeneration

Abstract

Hyperphosphorylation of the microtubule associated protein tau is detected in the brains of individuals with a range of neurodegenerative diseases including Alzheimer's disease (AD). An imbalance in phosphorylation and/or dephosphorylation of tau at disease-related sites has been suggested to initiate the abnormal metabolism and toxicity of tau in disease pathogenesis. However, the mechanisms underlying abnormal phosphorylation of tau in AD are not fully understood. Here, we show that the DNA damage-activated Checkpoint kinase 2 (Chk2) is a novel tau kinase and enhances tau toxicity in a transgenic Drosophila model. Overexpression of Drosophila Chk2 increases tau phosphorylation at Ser262 and enhances tau-induced neurodegeneration in transgenic flies expressing human tau. The non-phosphorylatable Ser262Ala mutation abolishes Chk2-induced enhancement of tau toxicity, suggesting that the Ser262 phosphorylation site is involved in the enhancement of tau toxicity by Chk2. In vitro kinase assays revealed that human Chk2 and a closely related checkpoint kinase 1 (Chk1) directly phosphorylate human tau at Ser262. We also demonstrate that Drosophila Chk2 does not modulate the activity of the fly homolog of microtubule affinity regulating kinase, which has been shown to be a physiological tau Ser262 kinase. Since accumulation of DNA damage has been detected in the brains of AD patients, our results suggest that the DNA damage-activated kinases Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in the pathogenesis of AD.

Figure 1.

Enhancement of human tau-induced retinal degeneration by overexpression of Drosophila Chk2. (A) Eye size is normal in a control fly with the pan-retinal gmr-GAL4 driver only. (B) Expression of human tau in the eye reduces eye size. (C) Flies co-expressing tau and Chk2 have smaller eyes than flies expressing tau alone. (D) Expression of Chk2 alone does not change eye size. (E) Expression of expanded polyglutamine repeat peptides (108 polyQ repeat peptides) in the eye causes depigmentation. (F) Expression of Chk2 does not enhance eye phenotype induced by expanded polyglutamine repeat peptides. (G) Expression of two copies of Alzheimer's amyloid β 42 peptides with Arctic mutation (50) in the eye causes degeneration. (H) Expression of Chk2 does not enhance eye degeneration induced by Alzheimer's amyloid-β 42 peptides with Arctic mutation.

Figure 2.

Tau phosphorylation levels increase at Ser262, but not at Ser202 or Thr231, when Chk2 is co-expressed in fly eyes. Fly heads expressing human tau alone (tau) or with Drosophila Chk2 (tau + Chk2) driven by the pan-retinal gmr-GAL4 driver were subjected to western blotting with anti-tau (human tau) or anti-phospho-tau antibodies. The phosphorylation levels at each site in fly heads co-expressing tau and Chk2 are normalized to the tubulin level and shown as a ratio relative to that in fly heads expressing tau alone. Asterisks indicate significant differences from control (n = 4 or 5, P < 0.05, Student's t-test). Representative blots are shown.

Figure 3.

The S262A tau mutation greatly reduces tau toxicity and abolishes Chk2-induced enhancement of tau toxicity. (A) Establishment of transgenic flies expressing human 0N4Rtau with the S262A mutation in fly eyes with the pan-retinal gmr-GAL4 driver. Western blot of head lysates with anti-human tau show that human wild-type tau and human S262A tau are expressed at similar levels in the transgenic flies. (B–E) External eyes of flies carrying the gmr-GAL4 driver only (B), expressing wild-type human tau (C), expressing S262A tau (D) or flies co-expressing S262A tau and Drosophila Chk2 (E).

Figure 4.

Checkpoint kinases directly phosphorylate human tau at Ser262 in vitro. Recombinant active human Chk2 (A and B) or recombinant active human Chk1 (C and D) were incubated with recombinant human tau in the presence or absence of inhibitors (Chk2 inhibitor II for Chk2 and UCN-01 for Chk1). Proteins were resolved by SDS-PAGE and visualized by western blotting using an anti-pSer262 antibody (A and C). The signals were quantified and the phosphorylation levels are shown as a ratio relative to that of recombinant tau incubated with recombinant kinase in the absence of inhibitor (B and D). Asterisks indicate significant differences (n = 4, P < 0.05, Student's t-test).

Figure 5.

Chk2 overexpression does not affect dMARK activity in fly eyes. A myc-tagged Drosophila dMARK was expressed alone or co-expressed with Drosophila Chk2 using the pan-retinal gmr-GAL4 driver. (A) Western blot with an anti-pT408 dMARK antibody (top) or an anti-myc antibody (bottom). The signals were quantified and the phosphorylation levels are shown as a ratio relative to that of flies expressing dMARK alone. No significant difference was detected (n = 4, P > 0.05, Student's t-test). Representative blots are shown. gmr: flies with the gmr-GAL4 driver only. The asterisk indicates a non-specific band. (B–D) External eyes from the gmr-GAL4 driver control (B), transgenic flies overexpressing dMARK alone (C) or flies co-expressing dMARK and Drosophila Chk2 (D). Chk2 overexpression did not enhance dMARK-induced eye degeneration.