Type of Document	Dissertation
Author	Felix, Augustine
URN	etd-04182011-000528
Title	Titin Kinase Interaction with Hax-1 in Nonmuscle Cells.
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Timothy M. Logan Committee Chair Geoffrey F. Strouse Committee Co-Chair Penny J. Gilmer Committee Member Thomas C. S. Keller III University Representative
Keywords	Y2H. hMSCs c-titin Hax-1 titin kinase Titin
Date of Defense	2011-03-30
Availability	unrestricted
Abstract Many non-muscle cells assemble actin-myosin II structures such as stress fibers to produce force for motility and adhesion. Stress fibers also serve as mechanosensors that signal changes in cell organization and gene expression. Many components of non-muscle cell stress fibers are known, but key aspects of stress fiber organization and signaling mechanisms remain poorly understood. Our lab discovered that isoforms of the multifunctional elastic protein titin exist in non-muscle cells and are localized in stress fibers. In striated muscles, titin plays important roles as a structural protein that assembles and maintains the structure of the sarcomere as well as a mechanosensor that signals changes in protein turnover and gene expression. It is likely that that cellular titin (c-titin) plays similar structural and regulatory roles in non-muscle cells. C-titin, like striated muscle titin, contains a kinase domain near its C-terminus, which might play a role in titin signaling activity. Yeast two hybrid (Y2H) screening of a HeLa cell cDNA library revealed that the c-titin kinase domain interacts with the C-terminal end of Hax1. Hax1 is a ubiquitously expressed multifunctional protein that interacts with a variety of proteins including the actin regulating protein, cortactin. Hax1 plays a variety of roles in modulating apoptosis and in regulating the contribution of actin cytoskeleton to cell adhesion and motility. Additional Y2H analysis using alanine mutants revealed that a -sheet cap region of the titin kinase domain interacts with a short highly conserved region of Hax1 encompassing residues 190-195 of the 279 residue protein. This titin kinase interaction region of Hax1 is near a possible phosphorylation site. Western blot analysis using a mouse monoclonal anti-serine/threonine primary antibody revealed that the titin kinase domain phosphorylates Hax1 in vitro. Immunolocalization revealed that some Hax1 and titin kinase domain colocalize in the lamellipodia of human mesenchymal stem cells. The interaction between the titin kinase domain and Hax1 points to a possible role for c-titin signaling in regulating actin cytoskeleton activity.
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