Figure 10.

Cdk5–p35 directly phosphorylates PIKfyve and regulates PI3,5P₂ levels. (A) pHA-p35 and pEGFP-Cdk5 were cotransfected into HEK293 cells. The Cdk5–p35 complex was immunoprecipitated using anti-HA antibody. Recombinant proteins: PIKfyve(1,700–2,098) and PIKfyve(1,214–1,594) were used for in vitro kinase assays. Red arrowheads indicate MBP-PIKfyve phosphorylated by Cdk5–p35 in vitro. Black arrowheads indicate MBP or MBP-PIKfyve peptides. (B) MEF labeled with [³H]inositol for 48 h and then treated with DMSO or 20 µM roscovitine for 3 h. Three independent experiments were performed. *, P = 0.014. (C) MEF labeled with [³H]inositol for 48 h, treated with DMSO or 20 µM roscovitine for 3 h, and then treated with high-salt media (final concentration 0.47 M NaCl; Salt) or without (Basal) for 30 min. Means ± SD are shown. n = 3. *, P < 0.005 by Student’s t test. PI, phosphatidylinositol. (D) Model for early protection via PI3,5P₂ signaling regulated by CDK5/Pho85 in response to the hyperosmotic stress. In hyperosmotic stress, a protection pathway activated by CDK5/Pho85 is distinct from the HOG pathway both spatially and temporally. Pho85–Pho80 directly activates the Fab1/PIKfyve complex. This activation is particularly crucial for the hyperosmotic stress–induced elevation of PI3,5P₂. These changes that signal from the vacuole/lysosome promote survival before the changes regulated by the HOG pathway, which signals from the nucleus.