Studies on the Activation Mechanism and Identification of aSubstrate for Giant Kinases in C. elegans Muscle Pubblico

Abstract

The muscles of virtually all animals contain giant (>700,000 Da) polypeptides that consist primarily of multiple copies of immunoglobulin (Ig) and fibronectin type III domains, and one or even two protein kinase domains. In various muscles these proteins have several roles. For example, directing assembly of the sarcomere and providing passive elasticity (titin of vertebrates), maintenance of the "catch" state (twitchin in mollusks), and ability of insect flight muscle to beat at high frequencies (projectin of insects). We are studying two such proteins in C. elegans, twitchin and TTN-1. Human titin kinase has been implicated as an initiating catalyst in a signaling pathway that ultimately results in muscle cell growth. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and a downstream autoinhibitory region. The precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Force- probe molecular dynamics simulations suggest that human titin kinase may act as a force sensor. This study predicts that the small forces that are generated with each contraction/relaxation cycle are sufficient to remove the autoinhibitory region thereby activating the enzyme. We experimentally tested this force activation hypothesis using atomic force microscopy to analyze the kinase and flanking domains of C. elegans TTN-1 (a titin-like protein) and twitchin. Our results show that these kinase domains have a remarkably high mechanical stability. Further, in response to applied force, these kinase domains unfold in a stepwise manner, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data directly support the hypothesis that the titin and titin-like kinase domains function as effective force sensors.

In an ongoing effort to identify binding partners and substrates for the protein kinase domains of these giants, we have discovered an excellent candidate for the TTN-1 protein kinase. The interacting partner is UIG-1, previously defined as an UNC-112 binding partner with Cdc42 GEF activity located in the dense bodies/I-bands of C. elegans striated muscle. An intragenic deletion of uig-1 displays disorganized myofibrils. Using the yeast 2-hybrid method we have determined which portions of each protein are required for this interaction, and we have confirmed this interaction using an in vitro binding assay. By immunofluorescence microscopy, UIG-1 partially co-localizes with TTN-1. Further, TTN-1 kinase phosphorylates UIG-1 in vitro in regions outside the DH and PH domains. We speculate that phosphorylation of UIG-1 by TTN-1 regulates either (1) the interaction of UIG-1 with UNC-112, (2) UIG-1's localization to dense bodies, or (3) the GEF activity of UIG-1. Whichever is the case, these studies have identified the first substrate for a muscle giant kinase in C. elegans and revealed a function for one of these kinases in sarcomere assembly.

Table of Contents

TABLE OF CONTENTS Chapter 1:

Muscle Structure: An Overview of Myofibrils.................................2 Caenorhabditis elegans as a Model for the Study of Muscle...............10 Titin and Titin-related Proteins..................................................16 Crystal Structures and Autoinhibition of Giant Protein Kinases............25 Molecular Force Spectroscopy..................................................36 Identification of Substrates for Protein Kinases..............................45 Rho Family of GTPases..........................................................48 Summary...........................................................................53 Chapter 2: Single Molecule Force Spectroscopy Reveals a Stepwise Unfolding of C. elegans Giant Protein Kinase Domains........................................55 Introduction........................................................................56 Results..............................................................................61 Discussion..........................................................................73 Materials and Methods............................................................81 Chapter 3: Identification and Characterization of the Substrate Interaction Between TTN-1 Kinase and UIG-1........................................................89 Introduction........................................................................90 Results..............................................................................92 Discussion........................................................................104 Materials and Methods..........................................................114

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular Biophysics, General
Parola chiave	twitchin single molecule atomic force microscopy molecular force spectroscopy kinase substrate giant protein kinase titin kinase C. elegans
Committee Chair / Thesis Advisor	Benian, Guy, Emory University
Committee Members	Wilkinson, Keith D, Emory University Dunlap, David, Emory University Lynn, David, Emory University Pallas, David C, Emory University Corbett, Anita, Emory University

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