Figure 1.
Differential dynamics of Cyto-Src and Lyn-Src activation by TNFα and IL1β.
(A) A schematic illustration of the structure and activation mechanism of the FRET-based Src biosensor. (B, C) The FRET ratio images and time courses of Cyo-Src activities (B) and Lyn-Src activities (C) under treatment with TNFα (gray) and IL1β (black). Color bars represent emission ratio of CFP/YFP of the biosensor, an index of Cyto-Src activation. The FRET ratio images were scaled according to the corresponding color bar. For each time-lapse imaging experiment, the images from the same cell were taken. The CFP/YFP emission ratios were averaged over the whole cell and were normalized to time 0. n = 8 (TNFα), 9 (IL1β) cells in (B); 6 (TNFα), 9 (IL1β) in (C). Scale bars, 10 µm. (D) The t1/2 values of Src response to TNFα and IL1β. * p<0.05 between Cyto-Src and Lyn-Src. (E) Gaussian function curves determined by curve fitting of the rate of mean FRET changes over time under cytokine treatment. (F, G) The normalized values of A, µ, and σ for Cyto-Src and Lyn-Src activities that were calculated by parameter fitting in cells under treatment with TNFα (F) or IL1β (G). n>6 cells. * p<0.05 between Cyto-Src and Lyn-Src. (H) The response of Src activities to cytokines in cells pretreated with CytoD (1 µg/ml, 1 h) to disrupt actin filaments or MβCD (10 mM, 1 h) to extract cholesterol from the plasma membrane. The Src activities at 2 hours after cytokine treatment were normalized to time 0. n>9 cells. * p<0.05 compared to the group treated with a corresponding cytokine alone.
Figure 2.
Salubrinal (Sal) and guanabenze (Gu) increase phosphorylation of eIF2α and decrease Cyto-Src activities.
(A) Western blots showing the elevated level of p-eIF2α by salubrinal and guanabenz. (B) Staining intensity of p-eIF2α, normalized by intensity of eIF2α. * p<0.05. (C) Cyto-Src activity by salubrinal and guanabenz. (D) Lyn-Src activity by salubrinal and guanabenz. Scale bars, 10 µm. n>7 cells.
Figure 3.
Salubrinal and guanabenz inhibit cytokine-induced Cyto-Src activity.
(A, B) C28/I2 cells transfected with either Cyto-Src or Lyn-Src biosensor were pretreated with TNFα or IL1β for 2 hours before incubating with salubrinal or guanabenz. (A) Effect of Salubrinal and guanabenz on cytokine-induced Cyto-Src activity. (B) Cytokine-induced Lyn-Src activity is not altered by salubrinal or guanabenz. Scale bars, 10 µm. n>7 cells.
Figure 4.
Involvement of eIF2α in salubrinal-driven Cyto-Src activity.
(A, B) C28/I2 cells were cotransfected with Cyto-Src or Lyn-Src biosensor, and eIF2α or NC siRNA, and then treated with 10 µM salubrinal for 1 hour during imaging. (A) eIF2α siRNA blocks inhibitory effect of salubrinal on Cyto-Src activity. (B) Lyn-Src activity is not altered by eIF2α siRNA. Scale bars, 10 µm. n>7 cells. (C) The basal level of Src activity in C28/I2 cells expressing NC or eIF2α siRNA. n>7 cells. * p<0.05 compared to the corresponding NC group.
Figure 5.
Fluid flow magnitude-dependent Lyn-Src activity.
(A) Selective Lyn-Src activities in response to different magnitudes of fluid flow. n>7 cells. (B) Cyto-Src activity is not altered by fluid flow. n>7 cells. (C, D) C28/I2 cells were cotransfected with either Lyn-Src or Cyto-Src biosensor and eIF2α siRNA or NC siRNA, and then subjected to fluid flow (10 dynes/cm2) during FRET imaging. (C) Lyn-Src activity under fluid flow. n>7 cells. (D) Cyto-Src activity under fluid flow. n>7 cells. (E) Lyn-Src activity in cytokine-treated cells under fluid flow(5 dynes/cm2). Cells transfected with a Lyn-Src biosensor were pretreated with cytokines for 2 hour before FRET imaging. n>7 cells. Scale bars, 10 µm.
Figure 6.
A proposed model of distinctive Src activities at different subcellular locations.
TNFα and IL1β activate Src kinases at the cytoplasm and lipid rafts of the plasma membrane, and actin cytoskeleton and lipid rafts are essential components of the Lyn-Src activation. Salubrinal can inhibit Src kinases in the cytoplasm through phosphorylation of eIF2α, but not in the lipid rafts of the plasma membrane. In contrast, fluid flow at 5 dynes/cm2 decreases integrin-mediated Src kinases in the lipid rafts of the plasma membrane, but it did not significantly affect the level of Src activation in the cytoplasm.