Abstract
Cell migration, a complex biological process, requires dynamic cytoskeletal remodeling. Phospholipase D (PLD) generates phosphatidic acid, a lipid second messenger. Although PLD activity has been proposed to play a role in cytoskeletal rearrangement, the manner in which PLD participates in the rearrangement process remains obscure. In this study, by silencing endogenous PLD isozymes using small interfering RNA in HeLa cells, we demonstrate that endogenous PLD1 is required for the normal organization of the actin cytoskeleton, and, more importantly, for cell motility. PLD1 silencing in HeLa cells resulted in dramatic changes in cellular morphology, including the accumulation of stress fibers, as well as cell elongation and flattening, which appeared to be caused by an increased number of focal adhesions, which ultimately culminated in enhanced cell-substratum interactions. Accordingly, serum-induced cell migration was profoundly inhibited by PLD1-silencing. Moreover, the augmented cell substratum interaction and retarded cell migration induced by PLD1-silencing could be restored by the adding back not only of wild type, but also of lipase-inactive PLD1 into knockdown cells. Taken together, our results strongly suggest that endogenous PLD1 is a critical factor in the organization of the actin-based cytoskeleton, with regard to cell adhesion and migration. These effects of PLD1 appear to operate in a lipase activity-independent manner. We also discuss the regulation of Src family kinases by PLD1, as related to the modulation of Pyk2 and cell migration.
Highlights
Cell motility is an integral part of a variety of signaling and cytoskeletal processes
Increased Number of Focal Adhesions and Enhanced Tyrosine Phosphorylation of Pyk2 by PLD1 Knockdown—Because phospholipase D (PLD) has been implicated in actin-based cytoskeletal rearrangement [17], and cellular morphology is generally regulated by cytoskeletal structures such as filamentous actin (F-actin) and Focal adhesion (FA), we evaluated the FA structures occurring in PLD1-knockdown cells by paxillin immunostaining
Pyk2 phosphorylation (Tyr-402) appears to be dependent on Src family kinases activity in these cells. This was confirmed by the fact that treatment with PP2, a specific inhibitor of Src family kinases, resulted in the complete abolition of Pyk2 phosphorylation. These results suggest that the enhanced phosphorylation of Pyk2 in the PLD1-knockdown cells was caused by the aberrant regulation of Src family kinases, and that the lipase-independent role of PLD1 is crucial for the proper regulation of these kinases
Summary
Cell motility is an integral part of a variety of signaling and cytoskeletal processes. PLD1 activity has been shown to be involved in lysophosphatidic acid-induced stress fiber formation in fibroblast cell lines [19]. These studies suggested possible roles for PLD catalytic activity in the actin-based cytoskeleton, many of the conclusions in these studies were predicated on the use of relatively nonspecific inhibitors, such as primary alcohols, or dealt with PLD overexpression, which can induce nonspecific or non-physiological cell responses. The silencing of PLD1 in HeLa cells induced dramatic changes in cell morphology, and increased Pyk and RhoA activation levels, resulting in increased focal adhesion formation. These changes were directly linked to the strength of cell-substratum interactions, and culminated in defective cell migration. The detailed molecular processes underlying these phenomena are not presented in this article, we suggest that Src family kinases are the vicinal targets of PLD1
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