VRK3-mediated nuclear localization of HSP70 prevents glutamate excitotoxicity-induced apoptosis and Aβ accumulation via enhancement of ERK phosphatase VHR activity

Most of neurodegenerative disorders are associated with protein aggregation. Glutamate-induced excitotoxicity and persistent extracellular signal-regulated kinase (ERK) activation are also implicated in neurodegenerative diseases. Here, we found that vaccinia-related kinase 3 (VRK3) facilitates nuclear localization of glutamate-induced heat shock protein 70 (HSP70). Nuclear HSP70 leads to enhancement of vaccinia H1-related phosphatase (VHR) activity via protein-protein interaction rather than its molecular chaperone activity, thereby suppressing excessive ERK activation. Moreover, glutamate-induced ERK activation stimulates the expression of HSP70 and VRK3 at the transcriptional level. Downregulation of either VRK3 or HSP70 rendered cells vulnerable to glutamate-induced apoptosis. Overexpression of HSP70 fused to a nuclear localization signal attenuated apoptosis more than HSP70 alone. The importance of nuclear localization of HSP70 in the negative regulation of glutamate-induced ERK activation was further confirmed in VRK3-deficient neurons. Importantly, we showed a positive correlation between levels of VRK3 and HSP70 in the progression of Alzheimer’s and Parkinson’s diseases in humans, and neurons with HSP70 nuclear localization exhibited less Aβ accumulation in brains from patients with Alzheimer’s disease. Therefore, HSP70 and VRK3 could potentially serve as diagnostic and therapeutic targets in neurodegenerative diseases.


Supplementary Figures
- Supplementary Fig. S1 Overexpression of FLAG-tagged VRK3 didn't affect the nuclear localization of HSP70 in SH-SY5Y cells under the normal conditions.
- Supplementary Fig. S2 Neither overexpression nor knockdown of VHR influences nuclear localization of HSP70 in glutamate-treated SH-SY5Y cells.
- Supplementary Fig. S3 The HSP70 F2 fragment containing the SBD was not sufficient to decrease glutamate-induced ERK activation.

Supplementary Tables
- Supplementary Table. S1 Clinicopathological details of subjects used for Western blotting -Supplementary Table. S2 Clinicopathological details of subjects used for immunohistochemistry Purification of fusion proteins. All GST-or His-tagged fusion proteins were expressed in Escherichia coli BL21 (DE3) pLysS (Novagen) and purified using glutathione-sepharose 4B agarose beads (GE Healthcare Bio-Sciences) or Ni-NTA agarose (Invitrogen) according to the manufacturer's instructions.
In vitro binding assay. For GST-pulldown assay, 3 µg of GST-tagged full length HSP70 and its fragments were incubated overnight with 2 µg of VHR in Triton lysis buffer containing 1 mM DTT and 10% glycerol, followed by incubation with glutathione-sepharose 4B beads (GE Healthcare Bio-Sciences) at 4 °C for 1 h. GST was used as negative control.
Immunoprecipitation. Cells were washed with chilled phosphate-buffered saline (PBS) and lysed for 30 min on ice using Triton lysis buffer supplemented with protease inhibitors (Roche). Lysates were sonicated and clarified by centrifugation at 15,000 rpm for 30 min. Immunoprecipitation was performed by incubating antibodies with lysates overnight followed by incubation with protein G beads (Roche) at 4 °C for 1 h.
After incubation on ice for 15 min, cellular debris was removed by centrifugation (3,000 rpm) at 4 °C for 5 min. Nuclei were resuspended and spun down twice in lysis/extraction buffer to avoid contamination of cytoplasmic proteins. To prepare nuclear extracts, pelleted nuclei were placed in nuclear extraction buffer containing 10 mM HEPES (pH 7.9), 0.1 mM EGTA, 1.5 mM MgCl 2 , 420 mM NaCl, 25% glycerol, 0.5 mM DTT, and 0.5 mM PMSF. After incubation on ice for 15 min, samples were sonicated and centrifuged (3,000 rpm) at 4 °C for 5 min. The supernatant was removed, and protein concentration was analyzed using Bradford reagent (AMRESCO).

Supplementary Methods
Assessment of cell viability and apoptosis. Cell viability was assessed using a chromogenic assay involving biological reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT), which is converted into blue formazan crystals by living cells. After electroporation with DNA or siRNA, cells were subcultured in 96-well plates at a density of 1×10 4 cells for 24 h in a final volume of 100 µl. Cells were then cultured in serum-free medium supplemented with glutamate for the indicated period of time. After the medium was removed, MTT (5 mg/ml in PBS) was added to the culture medium. Cells were incubated for 2 h in the dark at 37 °C, and the cell-free supernatant was removed from the wells. A total of MTT solvent (4 mM HCl and 0.1% Nondet P-40 [NP40], both in isopropanol) were added to dissolve the formazan crystal, and absorbance was measured at 570 nm using an ELISA reader with Infinite 200 Pro NanoQuant (TECAN). Apoptotic cells were further visualized using a modified TdT-mediated dUTP Nick End Labeling (TUNEL) assay with a DeadEnd Fluorometric TUNEL system (Promega) according to the manufacturer's protocol.