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 (PIP2) level increased rapidly in HeLa cells during hypertonic treatment. Depletion of the human type I phosphatidylinositol 4-phosphate 5-kinase beta isoform (PIP5KI beta) by RNA interference impaired both the PIP2 and actin cytoskeletal responses. PIP5KI beta 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 PIP5KI beta 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 PIP5KI beta dephosphorylation. Therefore, PIP5KI beta 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 PIP5KI beta dephosphorylation, lipid kinase activation, and PIP2 increase in cells. This PIP2 increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton.