Mitogen-activated protein kinases (MAPKs) are extremely important enzymes in signal transduction. The consequence of a breakdown in the normal control of these enzymes can lead to many devastating diseases such as cancer. Three major subfamilies have been identified in humans and long-term goals are to identify methods of inhibiting specific members of each subfamily. To achieve this goal a solid chemical-biology approach is taken, where enzymology and molecular biology is combined to establish fundamental properties of the enzymes. The first focus is to define the kinetic mechanism of the extracellular signal-regulated kinase, ERK2, the first MAPK to be discovered, and to test the hypothesis that ADP release is rate-limiting. Sophisticated pre-steady state quench-flow and stopped-flow fluorescence techniques, and equilibrium binding studies, will be used to quantify and identify individual enzymatic steps. The second focus builds on the kinetic model and tests the hypothesis that protein-protein interactions modulate ERK2 activity. A structural analysis-will identify ERK2-substrate interactions and substrate-induced conformational transitions with myelin basic protein and the truncated protein substrates c-Myc(1-100) and Ets-1 (1-138), using trace-labeling experiments. A powerful combination of site-directed mutagenesis and pre-steady state kinetics will critically probe the mechanistic implications of protein-protein interactions mediated by ERK2, focusing on a recently discovered modular binding domain found in many ERK2 substrates. The third focus will use peptide phage display technology to epitope-map ERK2-protein interactions and to test the hypothesis that protein-protein interactions mediated by ERK2 are driven by the recognition of small modular binding sequences that have evolved to form transiently stable complexes.
The specific aims during this period are: l) to define the kinetic mechanism of ERK2; 2) to perform structure-function studies to examine ERK2 substrate protein-protein interactions; and 3) to identify tight binding, modular peptide sequences by peptide phage display and contrast their function with wild type modular sequences. This will be the first comprehensive mechanistic study of ERK2 and the discovery of novel peptide inhibitors will set the scene for future structural and cell biology approaches aimed at understanding the breakdown in regulation of one of the primary enzymes in involved in human cancer and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059802-04
Application #
6727675
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
2001-04-01
Project End
2006-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
4
Fiscal Year
2004
Total Cost
$201,857
Indirect Cost
Name
University of Texas Austin
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712
Piserchio, Andrea; Warthaka, Mangalika; Kaoud, Tamer S et al. (2017) Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2. Proc Natl Acad Sci U S A 114:E6287-E6296
Tavares, Clint D J; Giles, David H; Stancu, Gabriel et al. (2017) Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION. J Biol Chem 292:2032-2045
Lee, Kwangwoon; Alphonse, Sébastien; Piserchio, Andrea et al. (2016) Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin. Structure 24:1441-51
Will, Nathan; Piserchio, Andrea; Snyder, Isaac et al. (2016) Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase. Biochemistry 55:5377-86
Zhan, Xuanzhi; Stoy, Henriette; Kaoud, Tamer S et al. (2016) Peptide mini-scaffold facilitates JNK3 activation in cells. Sci Rep 6:21025
Tavares, Clint D J; Devkota, Ashwini K; Dalby, Kevin N et al. (2016) Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening. Methods Mol Biol 1360:19-33
Piserchio, Andrea; Ramakrishan, Venkatesh; Wang, Hsin et al. (2015) Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2. Sci Rep 5:11127
Zhan, Xuanzhi; Kook, Seunghyi; Kaoud, Tamer S et al. (2015) Arrestin-3-Dependent Activation of c-Jun N-Terminal Kinases (JNKs). Curr Protoc Pharmacol 68:2.12.1-2.12.26
Ahmed, M Rafiuddin; Bychkov, Evgeny; Kook, Seunghyi et al. (2015) Overexpression of GRK6 rescues L-DOPA-induced signaling abnormalities in the dopamine-depleted striatum of hemiparkinsonian rats. Exp Neurol 266:42-54
Wang, Qiantao; Park, Jihyun; Devkota, Ashwini K et al. (2014) Identification and validation of novel PERK inhibitors. J Chem Inf Model 54:1467-75

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