Identification and Characterization of Cellular Titin

We previously discovered a large titin-like protein - c-titin - in chicken epithelial brush border and human blood platelet extracts that binds alpha-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells have revealed sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. These sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization reveals the majority of the c-titin colocalizes with alpha-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin which are incorporated with the actin stress fiber structures. Titin contains a dually regulated serine/threonine kinase domain belonging to the myosin light chain kinase family localized in the M-line region of titin. Titin kinase has a direct effect on myofibrillogenesis and protein turnover. Our lab has previously reported the identification of a novel nonmuscle titin isoform, cellular titin. Cellular titin localizes to the stress fibers of spread fibroblast and epithelial cells and contains sequence identical to the skeletal titin kinase domain. Expression of the cellular titin kinase domain in epithelial cells localize in a punctate manner previously reported, followed by a breakdown of stress fiber architecture resulting in cell rounding and eventual loss of adhesion to the substratum. Yeast two-hybrid analysis of the titin kinase domain has identified a novel binding partner HS1 associated protein X-1 (Hax-1). Previously identified as anti-apoptotic; its interaction with cortactin, Rac, and G[subscript α]13 illustrates Hax-1 as a mediator in stress fiber regulation and organization. Taken together our results suggest a novel role for cellular titin kinase in stress fiber integrity and regulation.