Phosphatidic acid has been found to be required for the recruitment of a particular Ras guanine nucleotide exchange factor, Sos, as well as Raf 1 to the plasma membrane. In a current study, we found that selective inhibition of choline kinase expression reduced phosphatidic acid and disrupted downstream MAPK and PI3K/AKT signaling. Given that CK37 decreased intracellular PF299804 molecular weight phosphatidic acid, we postulated that this compound also may disrupt signaling through PI3K/AKT and MAPK. while total ERK1/2 and AKT levels remained unchanged, as shown in Figure 3, experience of 10uM CK37 for 12 hours reduced initiating phosphorylations of ERK1/2 and AKT. Essentially, viability and cell phone number as of this early time point were identical between the vehicle control and CK37 exposure groups. CK37 Disrupts the Actin Cytoskeleton and Membrane Ruffling Phosphatidic acid has also been observed to promote actin polymerization, and these actin stress fibers have been shown to be necessary for prolonged MEK activation. pro-protein To analyze cytoskeletal arrangement in a reaction to CK37 treatment, we conducted immunofluorescence microscopy on HeLa cells using the small molecule phalloidin, which specifically binds to polymerized F actin, and an antibody for the focal adhesion protein vinculin. We found that, in the absence of CK37, HeLa cells exhibited comprehensive polymerization of F actin, which is straight anchored to the membrane at vinculin containing focal adhesion points. Nevertheless, incubation with 10uM CK37 interrupted the look of actin stress fibers in addition to the localization of focal adhesion points. Since CK37 changed the organization and was found to diminish the main lipid part of the cellular lipid bilayer, phosphatidylcholine, we investigated the effects of CK37 on the plasma membrane. Electron microscopy revealed ruffling and large membrane extensions in both HeLa and MDA MB 231 cells. However, incubation with 10uM ONX 0912 CK37 markedly attenuated these membrane structures, as apparent in Figure 4b. Transfection with the choline kinase siRNA caused the same disruption of the actin cytoskeleton and membrane ruffling as observed after CK37 exposure. These data support the the structural changes caused by CK37 might be directly related to the inhibition of choline kinase action caused by CK37. CK37 Selectively Reduces Cancer Cell Proliferation By Targeting Choline Kinase We examined the sensitivity of six neoplastic cell lines from both solid and hematologic roots to CK37 and found that incubation with CK37 caused a dose dependent suppression of cell growth in all six tumor cell lines. We next transiently transfected HeLa cells with a plasmid encoding the choline kinase open reading frame and examined the effects to the cytostatic activity of CK37.