Characterization of the Interaction Between Titn Kinase Domain and Enigma/Pdlim7

• Other Authors: Fazel, Arif (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Biology
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-4487

Titin is a very large protein that contributes to sarcomere structure and mechanosensing in striated muscle. Our lab discovered isoforms of titin in nonmuscle cells (Eilersten and Keller, 1992). Nonmuscle cell titin (c-titin) contains an alpha-actinin binding Z-repeat region, a distinctly PEVK region, a myosin filament-binding region, and the kinase domain also present in striated muscle titin isoforms. In striated muscle, the titin kinase domain (TKD) functions as a mechanosensor that signals changes in gene expression through interaction with nbr1 and p62. A previous yeast two hybrid (Y2H) screen to identify proteins that interact with the TKD in nonmuscle cells revealed an interaction with the ubiquitously expressed scaffold protein Enigma/PDLIM7. Enigma/PDLIM7 consists of an N-terminal PDZ domain that binds to β-tropomyosin on actin filaments, a Mid piece, and C-terminal region containing three LIM domains. The work described here further characterizes the interaction between the TKD and Enigma/PDLIM7. Y2H analysis with cloned TKD and Enigma/PDLIM7 fragments demonstrated that a region of the Enigma/PDLIM7 Mid piece and LIM1 and LIM3 domains interact with TKD. In vitro pull-down assays with bacterially expressed protein confirmed the interaction between TKD and Enigma LIM3. Immunolocalization of the TKD and Enigma/PDLIM7 in cultured human mesenchymal stem cells containing robust stress fibers revealed that both TKD and Enigma localized along stress fibers where they could interact, but there was little direct overlap in the cells under the standard culture conditions tested. These results support the possibility that Enigma/PDLIM7 functions as a scaffold to localize the TKD near actin filaments in the cytoskeleton of nonmuscle cells. === A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. === Summer Semester, 2011. === June 16, 2011. === Kinase, Enigma, Titin, Nonmuscle, Skeletal muscle === Includes bibliographical references. === Thomas C. S. Keller, III, Professor Directing Thesis; Thomas M. Roberts, Committee Member; Wu Min Deng, Committee Member.