Comparison of Protein Expression of the RhoA/ROCK Ca2+-Sensitization Pathway in both Cytosolic and Membrane Fractions of Murine Colon Smooth Muscle
AuthorDavidek, Nicole M.
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The RhoA/ROCK pathway has been targeted for study for the treatment of smooth muscle disorders, including gastrointestinal (GI) motility disorders. Since [Ca2+] in smooth muscle tissue initiates contraction, the RhoA/ROCK pathway allows for continual contraction at any level of Ca2+ within the cell. Using MYPT1, MYPT-pT696, MYPT1-pT853, LC20, pS19, and Rho kinase (ROCK 2), key components of RhoA/ROCK regulation of smooth muscle contraction, colon smooth muscle was analyzed for changes in MYPT1 and LC20 phosphorylation in both the membrane and cytosolic fractions. Carbachol (CCh), the Rho kinase inhibitor, SR-43677, and the PI3- kinase inhibitor, LY294002, were utilized to further determine RhoA/ROCK’s effect on MYPT1 and LC20 phosphorylation. Overall, higher levels of MYPT1, MYPT1-pT696 and pT853, and ROCK2 were found in the membrane fraction of the colon smooth muscle lysates. LC20 and pS19 were shown to have higher basal levels in the cytosolic fraction of the colon smooth muscle lysates. CCh caused an overall increase in the levels of MYPT1, MYPT1-pT696 and pT853, ROCK2, LC20, and pS19 with increasing exposure to CCh. Treatment with SR-43677 caused a decrease in MYPT1-pT853 in both fractions of the lysates, with MYPT1 showing a slight decrease and MYPT1-pT696 showing an increase in the pellet. LY294002 treatment did not have any significant effect on MYPT1, or MYPT1-pT853 or pT696, but overall caused a decrease in expression in the membrane fraction. Our results indicate that the RhoA/ROCK pathway is membranedependent, with its downstream effectors translocating to the membrane upon RhoA/ROCK activation.