Figure 8. Schematic illustration of PKR-mediated cell signaling that regulates cell migration. The activation or high expression of PKR results in the activation of p38 MAPK/MK2 which stimulates the phosphorylation of LIMK. The activated LIMK inhibits cofilin, resulting in the suppression of cell migration. doi:10.1371/journal.pone.0047721.g008 membrane. Cell membrane ruffling i.e. dynamic protrusion usually appears in the leading edge of a motile cell and is considered a dynamic parameter of cell motility [25]. An increase in membrane ruffling is positively associated with cell movement potential. To gain further insight into the relationship between PKR activation and cell motility, we investigated the effect of PIC on membrane ruffling using a time-lapse monitoring system. The cell membrane ruffling was recorded for 10 min in 10 second intervals. The kinetics of cell membrane ruffling was analyzed using kymography as previously described [14]. As shown in Fig. 7, PIC significantly decreased the rate of cell membrane ruffling. Both PKR inhibitor and SB203580 eliminated PIC-induced inhibition of membrane ruffling. These results further supported the findings presented above and indicated the PKR/p38 MAPK pathway regulated the motility of breast cancer cells. Discussion PKR has been implicated in anti-tumor action due to its anti- proliferative and pro-apoptotic potential. Here, for the first time, we demonstrate that activation of PKR inhibits the motility of breast cancer cells. PKR activation suppresses lamellipodia formation, cell spreading and membrane ruffling. We further established the p38 MAPK/MK2/LIMK/cofilin signaling path- way is involved in PKR modulation of cell migration (Fig. 8). PKR and Cancer Cell Motility PKR, the prototype of the eIF2a kinases, was first identified as an anti-viral serine/threonine kinase. Activation of PKR phos- phorylates eIF2a at Ser51, resulting in inhibition of protein synthesis. Apart from inhibition of protein synthesis, PKR activation can favor either the induction of cell cycle arrest or apoptosis [10]. Therefore, there is an increasing interest to explore PKR’s anti-tumor potential [3,7,10,26,27]. Available evidence supports the notion that activation of PKR can inhibit tumour cell Activation of PKR Inhibits Cell Migration growth and may have therapeutic benefits. It has been proposed to develop strategies of selective PKR activation in cancer cells to kill tumor cells [26]. PKR is over-expressed in some human cancers, such as breast cancer, melanoma cells and colon cancer [10]. Although the implication of high levels of PKR in these cancer cells is unclear, the high expression of PKR may offer an opportunity to target PKR and optimize cancer cell toxicity. Our study demonstrates that PKR activation inhibits the migration of breast cancer cells independent of cell cycle arrest and the induction of apoptosis, suggesting PKR activity may negatively regulate cell mobility. This finding is consistent with the report showing there is an inverse relationship between the aggressiveness and the expression/activity of PKR in breast cancer cells [28,29]. The current study focuses on breast cancer cells and shows that PKR activation impairs the migration of three aggressive breast cancer cell lines (BT474, MDA-MB231 and SKBR3). It remains to be determined whether the effect is general to all cell types. Cancer metastasis consists of multiple processes, and enhanced cancer cell motility is the hallmark of invasion and a critical step in each metastatic process [30]. Regardless whether PKR’s effect is cell type-specific or not, the novel finding provides a potential approach to developing strategies of cancer therapy targeting the PKR pathway, especially for those cancers overexpressing PKR. Signal Pathways that Mediate PKR Inhibition on Cell Motility Cell motility initiates with cell protrusion, i.e. lamellipodia formation which directs cell migration and is controlled by actin remodelling. We demonstrate that PKR activation inhibits lamellipodia formation, cell spreading and membrane ruffling, indicating a disruption of actin cytoskeleton reorganization is a mechanism of PKR activation-induced inhibition on cell motility. The cooperation between cofilin and Arp2/3 is essential for this initial step [31]. Cofilin severs actin filaments to create free barbed ends from which new filaments are elongated by Arp2/3. The force of actin depolymerization/polymerization on the leading edge drives cell protrusion. The distribution of active cofilin within leading edges of cells is a prerequisite for actin depolymerization/ polymerization which leads to cell migration. Alterations in overall activity of cofilin have been implicated in cancer metastasis and is directly associated with invasion and metastasis of mammary tumors [16,17]. The activity of cofilin is regulated by phosphor- ylation; phosphorylation of cofilin at Ser3 inactivates cofilin and abolishes its actin-severing ability, therefore inhibiting the formation of lamellipodia [23,32]. We demonstrate that PKR activation stimulates cofilin phosphorylation at Ser9, suggesting PKR activation impairs cell motility by inactivating cofilin (Fig. 8). Phosphorylation of cofilin at Ser3 is regulated by LIM kinase (LIMK) and testicular protein kinase [23,32]. Cofilin activity can also be regulated by dephosphorylation which is mediated by slingshot, chronophin and other phosphatases [17]. LIMK1 is activated through phosphorylation at Thr508 by downstream kinases of the Rho family GTPases, such as PAK, MRCK and ROCK [23,33–36]. Alternatively, LIMK1 can be activated by MAPK-activated protein kinase 2 (MK2), a substrate of p38 MAPK [24]. Overexpression of active LIMK1 inhibits the motility of mammary cancer cells [37]. We demonstrate that PKR activation causes the phosphorylation of p38 MAPK, MK2 and LIMK1 (Thr508), suggesting that this pathway is involved in PKR regulation of cofilin activity. PKR can activate p38 MAPK through interactions with mitogen-activated protein kinase kinase 6 (MKK6) [38–41]. Activation of p38 MAPK pathways is implicated in PKR activation-related immune response, apoptosis and cell cycle PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e47721