BRAF oncogenic mutations occur in approximately 8% of human cancers and lead to sustained activity of the Ser/Thr-specific protein kinases ERK1 and ERK2, which are critical mediators of cell entry into the S- phase of the human cell cycle. To begin to understand the principles governing substrate recognition and catalysis by ERK1/2, the structure and dynamics of the ERK27Ets1 (enzyme7substrate) complex will be determined in Aim 1, using an array of sophisticated NMR techniques. A semi-synthetic bisubstrate analog of transcription factor Elk-1, another substrate of ERK1/2, will be constructed to mimic phosphoryl transfer and analyzed in a similar manner. These studies will be complimented by X-ray crystal structure approaches.
In Aim 2, potent and highly selective Targeting Molecules that block interactions between ERK1/2 and its protein ligands will be acquired by greatly increasing the side chain diversity within consensus binding sequences. The mechanism of action and specificity of these targeting molecules will be determined using enzyme kinetics and structural approaches.
In Aim 3, the Targeting Molecules will be delivered into cells using protein transduction domains, and their efficacy determined with respect to their ability to inhibit Growth Factor-stimulated ERK1/2 activity and cell growth. Significantly, this multi-disciplined research program will provide potent and specific inhibitors of ERK1/2 for the first time and will allow ERK1/2 substrate-recruiting sites to be validated as potential drug targets for human cancers. The methodology developed herein will be rapidly applicable to other protein kinases, thereby potentially embracing many human diseases, and will provide the basis for future translational studies in pre-clinical mouse models.

Public Health Relevance

The focus of this application is an enzyme called ERK2 whose activity is upregulated in a large number of human cancers. The proposed studies will provide detailed structural information on how this enzyme interacts with other cellular proteins. A new technique will be developed to rapidly identify cell-permeable molecules that can prevent ERK2 from binding other cellular proteins. This will provide validation of ERK2 as a drug target, in models of human disease, and provide the experimental basis for validating other members of the same enzyme family. The studies proposed will be of high significance and will benefit human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059802-09
Application #
8130662
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
2001-04-01
Project End
2014-07-31
Budget Start
2011-08-01
Budget End
2014-07-31
Support Year
9
Fiscal Year
2011
Total Cost
$323,320
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, S├ębastien; 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
Devkota, Ashwini K; Edupuganti, Ramakrishna; Yan, Chunli et al. (2014) Reversible covalent inhibition of eEF-2K by carbonitriles. Chembiochem 15:2435-42

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