Cholinergic stimulation of arachidonic acid and phosphatidic acid metabolism in C62B glioma cells

Glioma C62B cells were incubated for 18 h with [1-14C]arachidonic acid. Most (80%) of the added [1-14C] arachidonic acid was taken into the intracellular pool; less than 1% of the intracellular [1-14C]arachidonic acid remained unesterified; the rest was present in glycerophospholipids. Acetylcholine stimulation of the prelabeled cells resulted in the rapid accumulation of free [1-14C]arachidonic acid, presumably liberated by hydrolysis from phospholipids. Labeled unesterified [1-14C]arachidonic acid peaked by 90 s and returned to basal levels by 5 min. Paralleling the transient increase of unesterified [1-14C]arachidonic acid were increases in level of radioactivity in an unidentified lipoxygenase metabolite of arachidonic acid and of radioactive phosphatidic acid. The release of arachidonic acid induced by acetylcholine or carbachol was blocked by muscarinic but not nicotinic receptor antagonists; adrenergic or histaminergic receptor agonists were ineffective at stimulating arachidonic acid liberation. In contrast to the transient effects of stimulation with cholinergic agonists, stimulation with the divalent cation ionophore A23187 resulted in a linear increase in the accumulation of liberated arachidonic acid for at least 1 h. Furthermore, the pattern of metabolites synthesized from arachidonic acid in response to ionophore stimulation was more complex than that observed following cholinergic stimulation and included also several metabolites derived from cyclooxygenase activity. We conclude that muscarinic receptor agonists rapidly induce specific changes in arachidonic acid and phosphatidic acid metabolism in a glioma cell line and suggest that similar responses may occur in glial cells and play a physiologically significant role in neural metabolism

Cortactin Phosphorylated by ERK1/2 Localizes to Sites of Dynamic Actin Regulation and Is Required for Carcinoma Lamellipodia Persistence

Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement.In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging.Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration