Myosin II‐A regulates the exocytosis of tumor necrosis factor receptor–1 (TNFR‐1)

TNF signaling through TNFR1 plays a critical role in cardiovascular functions and diseases. Relatively little is known about the intracellular trafficking of TNFR1, a critical event impacting the magnitude of the cellular response to TNF. Recent studies linking myosin II motor activities to the formation and trafficking of intracellular membrane vesicles prompted the hypothesis that myosin II motor activity has a role in regulating intracellular trafficking of TNFR1 to plasma membrane. Our measurement of the surface levels of TNFR1 in AT‐2 cardiomyocytes revealed that surface TNFR1 increases 10‐fold from a low basal state within 15 minutes of TNF binding. Inhibiting myosin II activation with either a myosin light chain kinase (MLCK) inhibitor, ML‐7; or over‐expression of a dominant negative, kinase dead MLCK abrogated myosin RLC phosphorylation and inhibited translocation of TNFR1 to the plasma membrane, resulting in attenuated TNF‐induced apoptosis. The use of a dominant negative, unphosphorylatable RLC to directly inhibit myosin II motor activity decreases cell surface levels of TNFR1 and greatly down‐regulates apoptosis response to TNF. Conversely activating myosin II using a phosphomimetic RLC results in elevated plasma membrane levels of TNFR1 even in unstimulated cells. In addition, pulse‐chase labeling of TNFR1 in AT‐2 and MDCK cells, fractionated on a discontinuous sucrose gradient, demonstrated that nonmuscle myosin II‐A is directly involved in the exocytosis of TNFR1‐containing vesicles from the Golgi‐apparatus to the plasma membrane. In conclusion, these studies demonstrate a novel role for myosin II motor activity in regulating TNFR1 signaling through regulating the exocytosis of TNFR1 to the plasma membrane in cardiomyocytes.