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The Dissertation Committee for Mark Allan Rainey Certifies That This Is the Approved Version of the Following Dissertation Copyright by Mark Allan Rainey 2004 The Dissertation Committee for Mark Allan Rainey certifies that this is the approved version of the following dissertation: A Structure/Function Analysis of Macromolecular Recognition by the Protein Kinase ERK2 Committee: Kevin Dalby, Supervisor Hung-wen Liu Andy Ellington John Tesmer Brent Iverson A Structure/Function Analysis of Macromolecular Recognition by the Protein Kinase ERK2 by Mark Allan Rainey, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December, 2004 Dedication To Mom and Dad Acknowledgements I would like to acknowledge Dr. Kevin Dalby for his help and support, Dr. Su Dharmawardhane for advice and support, and my committee members Dr. Andy Ellington, Dr. John Tesmer, Dr. Hung-wen Liu, and Dr. Brent Iverson for their discussions and review of this dissertation. I would also like to acknowledge the members of the Dalby laboratory for their support. September 2004 v A Structure/Function Analysis of Macromolecular Recognition by the Protein Kinase ERK2 Publication No. _________ Mark Allan Rainey, Ph.D. The University of Texas at Austin, 2004 Supervisor: Kevin N. Dalby Mitogen-activate protein kinases (MAPKs) phosphorylate protein substrates in the presence of magnesium and adenosine triphosphate in response to extracellular environmental signals to carry out signal-dependent intracellular responses. Extracellular signal-regulated protein kinase 2 (ERK2), a member of the MAPK family, mediates cellular growth, differentiation, and proliferation in response to growth factors. Understanding the mechanism by which MAPKs specifically recognize their protein substrates to carry out phosphoryl-transfer on specific residues within these macromolecules is critical for understanding the mechanism of signal transduction fidelity. Phage display was carried out against the active form of ERK2 to find novel ERK2-binding peptides. One peptide, KKKIRCIRGWTKDIRTLADSCQY, inhibited ERK2 phosphorylation of the protein substrate Ets∆138, exhibiting competitive and 2- mixed inhibition towards Ets∆138 (Ki = 20.7 ± 5.5 µM) and MgATP , respectively. vi Steady-state kinetics combined with a novel fluorescence anisotropy binding assay were used to quantitatively elucidate the roles of several proposed ERK2 exosites in forming a macromolecular docking complex with Ets∆138 required for efficient phosphorylation. An ERK2–Ets∆138 docking complex (Kd of 6.6 ± 1.2 µM) was shown to form independent of the substrate phospho-acceptor. Docking motif peptides proposed to bind ERK2 exosites could dissociate the ERK2–Ets∆138 docking complex, however, dissociation did not occur using a peptide containing an ERK2 phospho-acceptor indicating the lack of active site interactions in the docking complex. Mutation of ERK2 residues Lys-229 and His-230 to p38 MAPKα-like residues, an enzyme that does not efficiently phosphorylate Ets∆138, led to a 20-fold decrease in the specificity constant (kcat/Km) of Ets∆138 phosphorylation largely due to its inability to bind Ets∆138. This structure/function analysis offers a quantitative approach towards understanding the molecular determinants of protein substrate recognition by a protein kinase prior to phosphorylation. vii Table of Contents ABBREVIATIONS XV CHAPTER 1: MAPK SIGNALING 1 Signal Transduction Pathways........................................................................1 Specificity Determinants of Signal Transduction...........................................2 Mitogen-Activated Protein Kinases................................................................4 The Importance of Characterizing Protein-Protein Interactions.....................6 Elucidating Sites of MAPK Protein-Protein Interactions ........................................7 Computational Studies to Determine Functional Differences Between Subfamilies ............................................................................................7 Genetic Studies Reveal Sites of Interest .........................................................9 Yeast 2-Hybrid Studies.................................................................................10 Co-Crystallization of a Docking Motif Peptides and a MAPK ....................14 Hydrogen Exchange Mass Spectrometry......................................................16 Footprinting...................................................................................................20 Quantifying and Characterizing Protein-Protein Interactions................................22 Non-Quantitative Pull-Down Studies ...........................................................22 Isothermal Titration Calorimetry ..................................................................23 Surface Plasmon Resonance .........................................................................24 Fluorescence anisotropy/polarization ...........................................................25 The Function of MAPK Protein-Protein Interactions............................................27 Kinetic Studies of MAPK Catalysis .............................................................27 Enzymic MAPK Exosites .............................................................................28 MAPK-Interacting Docking Motifs..............................................................32 The DEF Docking Motif...............................................................................33 The DEJL Docking Motif .............................................................................34 A MAPKAPK/RSK-Like Docking Motif.....................................................35 Docking Mediates Enzymatic Specificity....................................................35 DEJL Motif Specificity.................................................................................36 Regulation of MAPK Docking Interactions .................................................36 viii Proline Binding Pocket .................................................................................37 Substrate Phosphorylation ............................................................................39 Highly Specific Enzymatic Interactions ................................................................40 MAPKK–MAPK Docking............................................................................40 MAPK–MKP Docking..................................................................................41 Non-Enzymatic Protein-Protein Interactions with MAPKs...................................43 Conclusions............................................................................................................45 CHAPTER 2: PHAGE DISPLAY IDENTIFIES NOVEL PEPTIDES THAT BIND ERK2 AND COMPETE WITH TRANSCRIPTION FACTOR BINDING 48 OVERVIEW ..........................................................................................................48 Purpose..........................................................................................................48 Approach.......................................................................................................48 Results...........................................................................................................50 Conclusions...................................................................................................50 INTRODUCTION .................................................................................................52 RESULTS AND DISCUSSION............................................................................55 Purification of Active ERK2.........................................................................55 Biotinylation of ERK2 ..................................................................................55 Immobilized b-ERK2 is Active ....................................................................57 Biopanning Scheme for Selecting Phage that Bind b-ERK2........................62 Titering Phage Stocks ...................................................................................64 Amplification of Phage Libraries..................................................................65 Round I: Non-Stringent Biopanning Conditions to Select Phage that Bind b- ERK2....................................................................................................65 Round II: Extending Wash Time to Select for Small koff Rates ...................67 Round III: Decreased Receptor Concentration to Encourage Phage Competition ..............................................................................................................70 Individual Phage Preparation........................................................................71 Phage Attachment Assay ..............................................................................72 Round IV: Decreased Receptor ....................................................................75 ix DTT Inhibits Cyclic Peptide Binding ...........................................................77 Ets∆138 Disrupts Phage Binding to b-ERK2 ...............................................79 MgATP2- Inhibits Phage Binding .................................................................80 Purification of Cyclic Peptide 20a ................................................................81 Inhibition of ERK2 by Cyclic Peptide 20a ...................................................84 Inhibition is Not Specific to ERK2...............................................................88
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