Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2011 Characterization of the Interaction Between Titn Kinase Domain and Enigma/Pdlim7 Arif Fazel Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES CHARACTERIZATION OF THE INTERACTION BETWEEN TITN KINASE DOMAIN AND ENIGMA/PDLIM7 By ARIF FAZEL A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science Degree Awarded: Summer Semester, 2011 The members of the committee approve the thesis of Arif Fazel defended on June 16, 2011 _________________________________ Dr. Thomas C.S. Keller III Major Professor _________________________________ Dr. Thomas M. Roberts Committee Member _________________________________ Dr. Wu Min Deng Committee Member Approved: ___________________________________________________ P. Bryant Chase, Chair, Department of Biological Science ___________________________________________________ Joseph Travis, Dean, College of Arts and Sciences The Graduate School has verified and approved the above-named committee members ii TABLE OF CONTENTS List of Tables……………………………………………………………………………..iv List of Figures…………………………………….……………………………………….v Abbreviations……....……………………………………………………………………..vi Abstract……...………………………………………………...…………………………vii 1. INTRODUCTION…………………….………………………………….……………1 2. MATERIALS AND METHODS……….…………………………………………….12 3. RESULTS..………………………………….………………….……………………..19 4. DISCUSSION……………………………………………………………….…….…..26 REFERENCES……………………………………………………………….………….30 BIOGRAPHICAL SKETCH…………………………………………………………….34 iii LIST OF TABLES Table 1. Primers for yeast vector pGAD-GH…………………………………………..14 Table 2. Primers for pET41a Protein expression vector…………………………….….16 Table 3. Sequences of Enigma………………………………………………………….17 Table 4. Table of Y2H results……………………………………………………….….21 iv LIST OF FIGURES Figure 1. Striated Sarcomere……………………………………………………..………2 Figure 2. Titin interactome……………………………………...………………..………2 Figure 3. Ribbon diagram of Titin Kinase Domain……………………...……….……...5 Figure 4. Organization of nonmuscle cell stress fiber…………………...……………….6 Figure 5. Diagram of Enigma protein…………………………………..…………..…...11 Figure 6a. GST-Enigma Mid Domain Purification………………………………….…..21 Figure 6b. GST-Enigma LIM domain purification……………………………….……..22 Figure 7. GST-Enigma LIM3 purification………………………………………………22 Figure 8. Gel Image of LIM3-Kin4 interaction…………………………….……….…..24 Figure 9. Immunofluorescent localization of TKK and PDLIM7 in hMSCs…….……..26 v ABBREVIATIONS Adaptor protein with PH and SH2 domain……………………...……………………..APS Enigma LIM domain 1……………………………………………………………….LIM1 Enigma LIM domain 2……………………………………………………………….LIM2 Enigma LIM domain 3……………………………………………………………….LIM3 Enigma Mid region ……………………………………………….……………….….MID Human mesenchymal stem cell…………………..………………………………….hMSC Insulin Receptor…………………………………………………….…………………InsR Lin-11, Isl-1, Mec-3…………………………….……...……………………….....…..LIM Oncogenic form of RTK…………………………………………….……………..Ret/ptc2 Postsynaptic density 95, Discs large, ZO-1……………………..………….…..…......PDZ . Receptor Tyrosine Kinases…………………………………………………....………RTK Serum Response Factor……………………………………...………….….……….....SRF Titin Kinase Domain………………………………………...………………..…….....TKD Yeast-2-Hybrid………………………………………………………………………..Y2H vi ABSTRACT 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. vii CHAPTER 1 GENERAL INTRODUCTION Striated Muscle Titin Striated muscle myofibers contain myofibrils composed of linearly arranged sarcomeres. The sarcomere is the functional unit of muscle contraction and where the three major filament proteins actin, myosin and titin interact. In sarcomeres thick filaments of myosin II, the molecular motor protein, and thin filaments of actin work together to drive the filament sliding that contracts muscle. The sarcomere is divided into four parts – the Z-line, I band, A-band and M-line. Actin filaments are anchored to the Z- line by interacting with the cross-linking protein α-actinin and extend into the I band. Myosin II thick filaments make up the A band and are anchored to the Z-line through titin and the M-line through an interaction with myomesin and titin. Titin is the third most abundant protein found in the sarcomere. Titin is a giant protein ranging in size from 2000 kDa to 4000 kDa depending on the muscle type. A single titin molecule is about 1 µm long and contains about 300 immunoglobulin (Ig) and fibronectin-like (Fn) domains. Each of these domains contains about 100 amino acids folded into a β-sheet sandwich. Single titin molecules span half the sarcomere, where the titin N-terminus is anchored to the Z-disc and the C-terminus anchored at the M-line (Figure 1). 1 Figure 1. Striated Sarcomere (Dr. F. Schoeni-Affoher, University of Fribourg, Switzerland. http://emedicine.medscape.com/article/1170911-overview#a0104) During muscle development, titin assists in the integration of the contractile proteins actin and myosin and is thought to act as a template directing assembly of thick filaments (Whiting, A. et al. 1989). Through the direct and indirect interactions that have been discovered to date, it is clear that titin functions as a scaffold that keeps the sarcomere intact (Figure 2). Further studies into titin are now demonstrating its involvement with muscle architecture, elasticity, and signaling. Figure 2. Titin interactome; direct (red) and indirect (yellow) interacting molecules of striated muscle titin. (Linke, W. 2008) 2 The Z-disk of striated muscle is one the most highly organized cellular structures. It anchors and aligns at least three major sarcomeric filament systems, including actin, titin and nebulin. Proper sorting and localization of proteins within the Z-disk region are critical for myofibril assembly, as well as for linking contractile functions of muscle sarcomeres to membrane systems. Parts of the Z-disk are also involved in signaling processes that may regulate muscle development and degradation. The titin N-terminus is anchored to the Z-disk through a series of protein-protein interactions. One is between the Z1Z2 domain of titin and telethonin. Studies show that titin interacting with telethonin is assembled into an antiparallel complex (Zou, P. et al. 2006). Downstream of the Z1Z2 region is the titin Z-repeat region that binds to α-actinin, which crosslinks titin with actin. The region of titin that spans the I-band between the Z-disc and the end of the myosin bipolar filament contains serially linked regions that confer passive elasticity in response to physiological stress (Horowits, R. et al. 1986). Passive tension in the I-band portion maintains the equidistance of the A-band between the Z-discs. The I-band region of titin is composed of tandem Ig domains, the N2-region, and a PEVK (Proline, Glutamate, Valine, Lysine) region. The Ig domain region of titin, located within the elastic section of this giant muscle protein, is responsible for the molecule’s extensibility and passive elasticity. The PEVK length in titin isoforms correlates with muscle stiffness and seems responsible for length gains at moderate sarcomere lengths where passive force increases, suggesting a role in muscle elasticity (Linke, W. 1998). The PEVK domain contains 110 to 225 exons, which encode 26 to 28 highly conserved amino acid domains, some of which are homologous to SH3 domains. The secondary and tertiary structure of the PEVK domains is not well understood. SH3-like domains are usually involved in signaling mechanisms. Based on these characteristics, it is suggested that the PEVK domain may serve as a mechanical and signaling stretch response center. The A-band region of titin is the largest with a molecular weight of 2 MDa and a length