Hypertonic stress increases phosphatidylinositol 4,5-bisphosphate levels by activating PIP5KIβ

Hyperosmotic stress increases phosphoinositide levels, reorganizes the actin cytoskeleton, and induces multiple acute and adaptive physiological responses. Here we showed that phosphatidylinositol 4,5-bisphosphate (PIP ₂) level increased rapidly in HeLa cells during hypertonic treatment. Depletion of the human type I phosphatidylinositol 4-phosphate 5-kinase β isoform (PIP5KIβ) by RNA interference impaired both the PIP₂ and actin cytoskeletal responses. PIP5KIβ was recruited to membranes and was activated by hypertonic stress through Ser/Thr dephosphorylation. Calyculin A, a protein phosphatase 1 inhibitor, blocked the hypertonicity-induced PIP5KIβ dephosphorylation/activation as well as PIP₂ increase in cells. Urea, which raises osmolarity without inducing cell shrinkage, did not promote dephosphorylation nor increase PIP₂ levels. Disruption or stabilization of the actin cytoskeleton, or inhibition of the Rho kinase, did not block the PIP₂ increase nor PIP5KIβ dephosphorylation. Therefore, PIP5KIβ is dephosphorylated in a volume-dependent manner by a calyculin A-sensitive protein phosphatase, which is activated upstream of actin remodeling and independently of Rho kinase activation. Our results establish a cause-and-effect relation between PIP5KIβ dephosphorylation, lipid kinase activation, and PIP₂ increase in cells. This PIP₂ increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton.