Studies of substrate specificity, regulation and inhibition of protein-tyrosine kinases

Persistent Link:
http://hdl.handle.net/10150/282141
Title:
Studies of substrate specificity, regulation and inhibition of protein-tyrosine kinases
Author:
Wu, Jinzi
Issue Date:
1996
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Protein tyrosine kinases (PTKs) play a crucial role in the regulation of cell proliferation, differentiation and signal transduction. However, substrate specificity and recognition motifs are not clear for most PTKs. Traditional methodologies for identifying substrate recognition motifs are often difficult and inefficient. In this dissertation, a novel approach for rapid discovery of linear substrate motifs of protein kinases has been developed using cyclic AMP-dependent protein kinase (cAPK) as a model system. This method is based on the screening of random synthetic combinatorial peptide libraries on beads where each bead expresses only one peptide entity. The peptide motif identified was RRXS, which exactly matched the motif for cAPK reported in the literature. Using this approach, a fraction of the random peptide library was screened for substrates of c-Src PTK. A heptapeptide, YIYGSFK, was identified and has been proven to be an efficient and specific substrate for c-Src PTK. A relatively specific pseudosubstrate-based peptide inhibitor of c-Src PTK has been developed based on the structure of YIYGSFK. Furthermore, a fraction of the octapeptide library was screened for substrates of c-Abl PTK. Fourteen peptides have been identified. Four different consensus sequences have been obtained by comparing the amino acid sequences of these peptides, suggesting that c-Abl PTK may have a relatively broad substrate specificity. Using these identified peptides as substrates, intrinsic tyrosine kinase activities of c-Abl and Bcr-Abl PTKs were compared. The data have suggested that Bcr sequences may directly activate the kinase activity of Abl protein. In addition, roles of the SH2 and SH3 domains in the regulation of the intrinsic kinase activity of Bcr-Abl have been studied. The results have indicated that the SH2 domain rather than SH3 domain was required for the intrinsic kinase activity of Bcr-Abl oncoprotein. In summary, the use of the combinatorial peptide library method has revealed some new insights on the substrate specificity and recognition motifs of PTKs, and suggested that small linear peptide motifs are important in the substrate recognition and phosphorylation by PTKs although the other factors may also contribute to the substrate specificity of PTKs.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Cell.; Chemistry, Biochemistry.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Cancer Biology
Degree Grantor:
University of Arizona
Advisor:
Lam, Kit S.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleStudies of substrate specificity, regulation and inhibition of protein-tyrosine kinasesen_US
dc.creatorWu, Jinzien_US
dc.contributor.authorWu, Jinzien_US
dc.date.issued1996en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractProtein tyrosine kinases (PTKs) play a crucial role in the regulation of cell proliferation, differentiation and signal transduction. However, substrate specificity and recognition motifs are not clear for most PTKs. Traditional methodologies for identifying substrate recognition motifs are often difficult and inefficient. In this dissertation, a novel approach for rapid discovery of linear substrate motifs of protein kinases has been developed using cyclic AMP-dependent protein kinase (cAPK) as a model system. This method is based on the screening of random synthetic combinatorial peptide libraries on beads where each bead expresses only one peptide entity. The peptide motif identified was RRXS, which exactly matched the motif for cAPK reported in the literature. Using this approach, a fraction of the random peptide library was screened for substrates of c-Src PTK. A heptapeptide, YIYGSFK, was identified and has been proven to be an efficient and specific substrate for c-Src PTK. A relatively specific pseudosubstrate-based peptide inhibitor of c-Src PTK has been developed based on the structure of YIYGSFK. Furthermore, a fraction of the octapeptide library was screened for substrates of c-Abl PTK. Fourteen peptides have been identified. Four different consensus sequences have been obtained by comparing the amino acid sequences of these peptides, suggesting that c-Abl PTK may have a relatively broad substrate specificity. Using these identified peptides as substrates, intrinsic tyrosine kinase activities of c-Abl and Bcr-Abl PTKs were compared. The data have suggested that Bcr sequences may directly activate the kinase activity of Abl protein. In addition, roles of the SH2 and SH3 domains in the regulation of the intrinsic kinase activity of Bcr-Abl have been studied. The results have indicated that the SH2 domain rather than SH3 domain was required for the intrinsic kinase activity of Bcr-Abl oncoprotein. In summary, the use of the combinatorial peptide library method has revealed some new insights on the substrate specificity and recognition motifs of PTKs, and suggested that small linear peptide motifs are important in the substrate recognition and phosphorylation by PTKs although the other factors may also contribute to the substrate specificity of PTKs.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Cell.en_US
dc.subjectChemistry, Biochemistry.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineCancer Biologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLam, Kit S.en_US
dc.identifier.proquest9713351en_US
dc.identifier.bibrecord.b34328993en_US
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