2 and actin cytoskeletal responses. PIP5KI␤ 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␤ dephosphorylation/activation as well as PIP 2 increase in cells. Urea, which raises osmolarity without inducing cell shrinkage, did not promote dephosphorylation nor increase PIP 2 levels. Disruption or stabilization of the actin cytoskeleton, or inhibition of the Rho kinase, did not block the PIP 2 increase nor PIP5KI␤ dephosphorylation. Therefore, PIP5KI␤ 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␤ dephosphorylation, lipid kinase activation, and PIP 2 increase in cells. This PIP 2 increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton.All cells experience fluctuations in osmolarity. Unicellular organisms and plants continuously confront osmotic challenges in their environment. In higher animals, the kidney and the gastrointestinal system are routinely exposed to severe osmotic fluctuation, while the majority of cells in other organs are protected from large tonicity changes. Nevertheless, these other organs are also confronted with transient osmolarity variations due to changes in the transmembrane transport of solutes or shifts in the balance between low molecular weight precursors and their macromolecular products. Recently, there has been a renewed interest in understanding the mechanism of hypertonic response in the clinical arena (1), due to the discovery that treatments using hypertonic resuscitation in experimental models of trauma, hemorrhagic shock, sepsis, and burn injury are more beneficial than conventional isotonic resuscitation (2, 3). While the fundamental mechanism for such protection is not completely understood, the actin cytoskeleton, which is reorganized during hypertonic stress, has been implicated (3, 4).Actin remodeling as well as many of the other hyperosmotic responses are evolutionary conserved. These include large shifts in phosphoinositide metabolism, activation of the mitogen-activated protein and tyrosine kinase pathways, volume regulation and the reprogramming of gene transcription (3, 5). Phosphatidylinositol 4-phosphate (PI4P) 4 and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) levels increase dramatically in mammalian cardiac muscle and tissue culture cells that were exposed to hypertonic sucrose or NaCl (6). Other phosphoinositides, including phosphatidylinositol 3,5-bisphosphate (7, 8), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate are increased in some other types of cells as well (9).Hyperosmotic stress acutely induces cell shrinkage, which is subsequently corrected by volume regulation. The response cascade can be classified into four mecha...

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) has many essential functions and its homeostasis is highly regulated. We previously found that hypertonic stress increases PIP 2 by selectively activating the ␤ isoform of the type I phosphatidylinositol phosphate 5-kinase (PIP5K␤) through Ser/Thr dephosphorylation and promoting its translocation to the plasma membrane. Here we report that hydrogen peroxide (H 2 O 2 ) also induces PIP5K␤ Ser/Thr dephosphorylation, but it has the opposite effect on PIP 2 homeostasis, PIP5K␤ function, and the actin cytoskeleton. Brief H 2 O 2 treatments decrease cellular PIP 2 in a PIP5K␤-dependent manner. PIP5K␤ is tyrosine phosphorylated, dissociates from the plasma membrane, and has decreased lipid kinase activity. In contrast, the other two PIP5K isoforms are not inhibited by H 2 O 2 . We identified spleen tyrosine kinase (Syk), which is activated by oxidants, as a candidate PIP5K␤ kinase in this pathway, and mapped the oxidant-sensitive tyrosine phosphorylation site to residue 105. The PIP5K␤Y105E phosphomimetic is catalytically inactive and cytosolic, whereas the Y105F non-phosphorylatable mutant has higher intrinsic lipid kinase activity and is much more membrane associated than wild type PIP5K␤. These results suggest that during oxidative stress, as modeled by H 2 O 2 treatment, Syk-dependent tyrosine phosphorylation of PIP5K␤ is the dominant post-translational modification that is responsible for the decrease in cellular PIP 2 .