Figure 7

ERK and ROCK-mediated regulation of myosin light chain phosphatase during TMV shedding.

(A) Untransfected cells or those transfected with T7-tagged RhoA(G14V) were grown on a thick gelatin matrix and treated with either 50 μM NSC23766 (NSC), 20 μM U0126 (U), or both, prior to western blotting for total and phospho-MLC^18/19, total and phospho-ERK^202/204, the T7 tag and α-tubulin. (B) Untransfected cells and those transiently transfected with RhoA(G14V) were grown on a thick FITC-gelatin matrix and treated with 20 μM U0126 prior to fixation and staining to visualize β₁ integrin (red). (C) Control cells and those transiently transfected to express RhoA(G14V) were grown on a thick FITC-gelatin matrix and treated with U0126, ML-7, or NSC23766, as indicated prior to fixation and staining to visualize β₁ integrin and the T7-tagged mutants. Released microvesicles were quantified as described in the Methods and the error bars represent the standard error of the mean. (D) Cells were grown on thick gelatin matrix and treated with U0126, Y-27632, NSC23766, or a combination thereof as indicated, prior to western blotting to assess phospho-MYPT1 Thr⁶⁹⁶, phospho-MYPT1 Thr⁸⁵³, phospho-ERK^202/204, total ERK and α-tubulin. (E) LOX, LOX^ARF6-GDP, LOX^ARF6-GTP and LOX^ARF6-GTP treated with 50 μM NSC23766 (NSC) were grown on a thick gelatin matrix prior to lysis and western blotting for total and phospho-MYPT1⁸⁵³ and α-tubulin. (F) Schematic representation of signaling pathways that lead to MLC phosphorylation to promote TMV shedding. In addition to the ARF6-ERK-MLCK pathway previously described¹⁷, RhoA signaling also promotes TMV shedding by activation of ERK and ROCK leading to the inhibitory phosphorylation of the MYPT1 subunit of myosin phosphatase. ARF6 activation and Rac1 inhibition feed into pathways that promote RhoA activation. All western blot film images were cropped to show the proteins of interest. All protein gels were run using 12% acrylamide, except for MYPT1 blots which were run using 8% acrylamide.