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dc.contributor.advisorDalby, Kevin N.en
dc.creatorRainey, Mark Allanen
dc.date.accessioned2008-08-28T21:59:00Zen
dc.date.available2008-08-28T21:59:00Zen
dc.date.issued2004en
dc.identifierb59340551en
dc.identifier.urihttp://hdl.handle.net/2152/1392en
dc.descriptiontexten
dc.description.abstractMitogen-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 mixed inhibition towards Ets∆138 (Ki = 20.7 ± 5.5 µM) and MgATP2-, respectively. 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.
dc.format.mediumelectronicen
dc.language.isoengen
dc.rightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.en
dc.subject.lcshProtein kinase ERK2en
dc.subject.lcshProtein bindingen
dc.titleA structure/function analysis of macromolecular recognition by the protein kinase ERK2en
dc.description.departmentInstitute for Cellular and Molecular Biologyen
dc.identifier.oclc58389500en
dc.identifier.proqst3150715en
dc.type.genreThesisen
thesis.degree.departmentCellular and Molecular Biology, Institute foren
thesis.degree.disciplineMolecular Biologyen
thesis.degree.grantorThe University of Texas at Austinen
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen


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