The goal of this grant is to elucidate the physiological role of tropomyosin (TM) and caldesmon in the regulation and function of actomyosin contractility in nonmuscle cells. The six to eight TM isoforms found in normal fibroblasts are an integral part of the microfilament system, although it is still poorly understood how the different isoforms contribute to the regulation of myosin II function. Furthermore, activation of actomyosin contractility is an essential step during adhesion-dependent signaling and the subsequent generation of intracellular signals required for cell proliferation. Most studies of the regulation of contractility in nonmuscle cells and its relationship to adhesion-dependent signaling have focused on the role of myosin light chain phosphorylation and its upstream regulators. The role played by specific TM isoforms and caldesmon in the regulation of nonmuscle contractility and adhesion-dependent signaling remains to be carefully analyzed. Changes in cellular proteins involved in the regulation of myosin II function observed in transformed cells might lead to increases in actomyosin contractility and the corresponding downstream signaling pathways that contribute to aberrant growth.
In Specific Aim 1, biochemical assays will be used to examine how the interaction of specific TMs along actin filaments affects the function of myosin II and the actions of other regulatory proteins, such as caldesmon.
Specific Aim 2 will address how alterations in the expression of TM in vivo effect cellular contractility and adhesion-mediated signaling. The experiments in Specific Aim 3 will examine if caldesmon is a target for phosphorylation in normal and transformed fibroblasts and how this phosphorylation affects cellular contractility and adhesion-mediated signaling. These studies will provide important new information regarding the role of these actin-filament associated proteins in the regulation of myosin II function and adhesion-dependent signaling, and the potential of inhibiting signal transduction pathways dependent on actomyosin contractility as a therapeutic target and adjuvant for the treatment of cancer.
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