166 research outputs found

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

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    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

    Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets.

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    Thrombin activates phospholipase C in human platelets, but the specific isoenzymes activated and the signal pathway used are unknown. Using specific antibodies, we found that phospholipase C-gamma 1 and the p21ras GTPase-activating protein, rasGAP, are present in human platelets. Furthermore, phospholipase C-gamma 1 was detectable, based on enzyme activity and Western blot analysis, in immunoprecipitates of rasGAP, suggesting that these two proteins form tight complexes. The pool of phospholipase C-gamma 1 associated with rasGAP was phosphorylated but not through tyrosine phosphorylation. Although thrombin stimulation had no effect on the level of phosphorylation of phospholipase C-gamma 1 and only slightly increased the tyrosine phosphorylation of rasGAP, the agonist induced the association of rasGAP with rap1B, as indicated by the appearance of rap1B on a Western blot of rasGAP immunoprecipitates. Our results suggest the formation of a signaling complex involving rasGAP, phospholipase C-gamma 1, and rap1B that might be important in the cascade leading to platelet activation
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