Role of Cellular Titin in Cytoskeletal Function

MCB 9507003托马斯凯勒III 肌球蛋白II和肌动蛋白丝在细胞中的相互作用为包括胞质分裂在内的重要细胞过程产生力。肌球蛋白II仅限于与肌动蛋白丝的子集相互作用的机制，如在细胞的某些区域或结构，如应力纤维和卵裂沟仍然知之甚少。在脊椎动物横纹肌中，肌球蛋白II细丝通过与肌联蛋白相互作用而被拴在肌节结构上。除了结构上的作用外，肌肉肌联蛋白还可以通过在脊椎动物肌肉肌联蛋白和无脊椎动物肌肉微小肌联蛋白中发现的激酶结构域的活性在肌节组装和收缩中发挥调节作用。 PI发现了c-titin，一种与肠上皮细胞刷状缘细胞骨架和其他细胞的应力纤维和卵裂沟中的细胞肌球蛋白II共定位的肌球蛋白的细胞同种型，提出了c-titin在定位和组织细胞肌球蛋白II中发挥作用的可能性。另外的支持来自于证明，c-titin可以在体外将细胞肌球蛋白II细丝组织成高度有序的阵列，并且平滑肌α-辅肌动蛋白和肌动蛋白并入c-titin-肌球蛋白II结构中以产生类似于应力纤维的组装。肌动蛋白-肌球蛋白系统的组装依赖于肌动蛋白相关蛋白α-辅肌动蛋白和肌球蛋白II相关蛋白c-肌联蛋白之间的直接相互作用。 本研究的目的是进一步研究c-titin的特性，可能有助于其功能。第一个具体的目的是使用纯化的蛋白质在体外重组试验中重新研究的相互作用的α-辅肌动蛋白与c-titin使用细胞亚型的α-辅肌动蛋白的相互作用与肌动蛋白是Ca 2+敏感的。本研究将揭示细胞α-辅肌动蛋白和c-titin之间的相互作用是否也是Ca ~（2+）敏感的，从而阐明Ca ~（2+）如何调节肌动蛋白-肌球蛋白II细胞骨架结构的稳定性和整合。第二个具体目的是确定c-肌联蛋白是否具有调节c-肌联蛋白或其他蛋白质活性的激酶活性。c-titin自磷酸化的能力表明，它具有与其他充分表征的激酶（包括CaM激酶II）相似的酶促机制和活性调节手段。还将测试其他几种蛋白质作为c-肌联蛋白激酶活性的可能底物，包括肌球蛋白重链和轻链。证明c-肌联蛋白激酶活性和确定可能的底物将支持c-肌联蛋白在细胞骨架中发挥更多结构作用的可能性。第三个具体目标将解决c-titin是否是其他激酶的底物，从而受其他激酶调节，包括cAMP依赖性蛋白激酶和蛋白激酶C。将测试各种激酶的体外磷酸化对体外组织肌球蛋白丝中c-肌联蛋白活性的影响。这三个生化特性应阐明一个重要的新的细胞骨架组织细胞的组成部分的关键特性。 %细胞使用肌球蛋白来产生重要细胞功能的力量，如细胞分裂。是什么控制肌球蛋白的定位，从而在细胞中哪里可以产生力仍然是一个谜。肌联蛋白，一种新发现的肌球蛋白相关蛋白的活性的调查，应阐明肌球蛋白活性的细胞调节的关键方面。 ***

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. ***