| Abnormal regulation of intracellular signaling proteins may lead to uncontrolled cell growth and alterations in cellular adhesions, engendering tumorigenesis and metastasis, respectively. The extracellular-related kinase (ERK) family of mitogenic-activated protein kinases (MAPKs) is comprised of serine/threonine kinases involved in mitogenic signaling in response to growth factors, hormones, and other extracellular signals. Numerous signaling pathways result in ERK activation, and their delineation is complicated by the copious overlap that exists between agonist pathways that regulate ERK activity. In this study, we examined regulation of ERK signaling cascades in Rat-1 HIR fibroblasts, EL4 thymoma cells that exhibit phorbol ester-sensitive and -resistant phenotypes, and EL4 cells stably transfected with phospholipase D2 (PLD2), and enzyme postulated to be involved in regulation of ERK activity.; Our results indicated that in Rat-1 HIR fibroblasts, insulin and phorbol 12-myristate 13-acetate (PMA) synergistically activate ERK via a mechanism that may involve Shc and/or focal adhesion kinase (FAK), but does not appear to involve PLD activation. In EL4 cells, FAK is not expressed in PMA-sensitive cells that exhibit rapid ERK activation in response to PMA, but is expressed in PMA-resistant cells that exhibit decreased and delayed ERK activation in response to PMA. PMA-sensitive and -resistant EL4 cells also vary in their expression of Pyk2, a FAK-related protein, and RasGRP, a protein implicated in ERK activation. Transient transfection studies suggest that Pyk2 and RasGRP contribute to PMA-induced ERK activation in EL4 cells. Stable expression of human PLD2 in PMA-resistant EL4 cells fails to enhance PMA-stimulated ERK activation, but results in enhanced cellular adhesion and activation of FAK. These results highlight and provide insight into the complex events regulating ERK activity, and suggest a novel role for PLD2 in FAK activation and cellular adhesion. |
