Activation of PKR Inhibits Cell Migration Figure 7. Activation of PKR inhibits dynamic cell membrane ruffling. A: MDA-MB231 cells were pretreated with DMSO (control), PKR-I (500 nM) or SB203580 (5 mM) for indicated hours, then followed by the treatment of PIC (10 ng/ml) for 6 hours. Top panel: Phase-contrast frames were acquired every 10 seconds for 10 minutes on a time-lapse microscope using a 60X oil-immersion lens. Scale bar = 10 mm. Bottom panel: The corresponding kymographs of the movie generated along a line transecting the cell membrane on the lamellipodia are presented. B: Relative rate of cell membrane ruffling was calculated from kymographs. The experiments were replicated five times. * denotes a statistically significant difference from all other groups (p,0.05). doi:10.1371/journal.pone.0047721.g007 leading edge is required for the initiation of cell movement [22], PIC-induced inhibition of cofilin activity and redistribution of cofilin may account for decreased lamellipodia formation and cell movement. We next examined the potential signal pathways that regulate cofilin activity. LIM kinase 1 (LIMK1) is an important upstream kinase of cofilin that regulates the phosphorylation of cofilin [23]. LIMK activity is subjected to the regulation of p38 MAPK/MK2 pathways [24]. As shown in Fig. 4A, PIC induced phosphorylation of PKR (Thr446), LIMK (Thr505/508), p38 MAPK (Thr180/183), MK2 (Thr334) and cofilin (Ser3). PKR inhibitor blocked PIC-induced phosphorylation of p38 MAPK and MK2 (Figs. 2A and B), but the p38 MAPK inhibitor (SB203580) did not affect PKR phosphorylation (Figs. 5A and B), suggesting that PKR is upstream of p38 MAPK and MK2. Next, we sought to determine whether PIC-induced cofilin phosphorylation was mediated by p38 MAPK. As shown in Figs. 5A and B, SB203580 diminished PIC-induced phosphor- ylation of p38 MAPK, MK2, LIMK as well as cofilin. The finding was confirmed by immunofluorescent staining (Fig. 5C). These results suggested that p38 MAPK was upstream of LIMK and cofilin; PIC-mediated activation of p38 MAPK resulted in phosphorylation of LIMK and cofilin. Furthermore, SB203580 alleviated PIC-induced inhibition of cell migration (Fig. 5D). The involvement of PKR in p38/MK2 signaling was further supported by the study using PKR siRNA. As shown in Fig. 6, treatment of PKR siRNA blocked PIC-induced activation of p38/MK2. Taken together, these results suggested the activa- tion of PKR suppressed cofilin activity through activation of the p38/MK2/LIMK pathway. Activation of PKR Inhibits Cell Membrane Ruffling Cell membrane ruffling is a dynamic movement with rapid irregular vacillation of protrusion and withdrawal of a cell surface PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47721