MCB9507003 Thomas Keller III The interaction of myosin II and actin filaments in cells produces force for important cell processes including cytokinesis. The mechanism by which myosin II is restricted to interacting with only subsets of actin filaments such as those in certain regions of cells or in structures such as stress fibers and cleavage furrows remains poorly understood. In vertebrate striated muscle, myosin II filaments are tethered to the sarcomere structure by interaction with muscle titin. Besides a structural role, muscle titins may also play regulatory roles in sarcomere assembly and contraction through activity of a kinase domain found in vertebrate muscle titins and invertebrate muscle minititins. The PI's discovery of c-titin, a cellular isoform of titin that is colocalized with cellular myosin II in the intestinal epithelial cell brush border cytoskeleton and in stress fibers and cleavage furrows of other cells, raised the possibility that c-titin plays a role in localizing and organizing cellular myosin II. Additional support comes from the demonstrations that c-titin can organize cellular myosin II filaments into highly ordered arrays in vitro and that smooth muscle alpha-actinin and actin incorporate into the c-titin-myosin II structures to yield assemblies that resemble stress fibers. Assembly of the actin-myosin system depends on a direct interaction between a-actinin, an actin-associated protein, and c-titin, a myosin II-associated protein. The objective of the research is to further investigate properties of c-titin that could contribute to its function. The first specific aim is to use purified proteins in in vitro reassembly assays to reinvestigate the interaction of alpha-actinin with c-titin using a cellular isoform of alpha-actinin for which interaction with actin is Ca2+-sensitive. This investigation will reveal whether the interaction between cellular a-actinin and c-titin also is Ca2+-sensitive thus elucidating how Ca2+ might regulate stability and integra tion of actin-myosin II cytoskeletal structures. The second specific aim is to determine whether c-titin has kinase activity that regulates the activity of c-titin or other proteins. The ability of c-titin to autophosphorylate would suggest that it has an enzymatic mechanism and means of activity regulation similar to those of other well-characterized kinases, including CaM kinase II. Several other proteins also will be tested as possible substrates for c-titin kinase activity, including myosin heavy and light chains. Demonstrating c-titin kinase activity and identifying possible substrates will support the possibility that c-titin plays more than a structural role in cytoskeletons. The third specific aim will address whether c-titin is a substrate for and thereby regulated by other kinases, including cAMP-dependent protein kinase and protein kinase C. In vitro phosphorylation by various kinases will be tested for effect on c-titin activity in organizing myosin filaments in vitro. These three biochemical characterizations should elucidate key properties of an important new cytoskeletal organizing component of cells. %%% Cells use the protein myosin to produce force for important cellular functions such as cell division. What controls the localization of myosin and thus where force can be produced in cells remains a mystery. Investigation of the activity of c-titin, a newly identified myosin associated protein, should elucidate key aspects of cellular regulation of myosin activity. ***
