Use of the Traceable Kinase Method to identify protein substrates that mediate PKCalpha-stimulated motility in human breast cells.
Item
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Title
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Use of the Traceable Kinase Method to identify protein substrates that mediate PKCalpha-stimulated motility in human breast cells.
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Identifier
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AAI3295017
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identifier
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3295017
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Creator
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Abeyweera, Thushara Preeni.
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Contributor
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Adviser: Susan A. Rotenberg
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Date
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2007
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Identification of the proteins that are phosphorylated by PKCalpha posed a major challenge until the introduction of the Traceable Kinase Method. This approach consists of engineering the ATP binding site of PKCalpha so that it can accommodate a larger analogue of ATP, such as N6-phenyl-ATP, that cannot be bound by the wildtype PKCalpha or presumably any other protein kinase. [gamma-32P]-N 6-phenyl-ATP was used by the mutant PKCalpha to radiolabel substrate proteins that co-immunoprecipitated with mutant PKCalpha from a total human breast cell lysate. Following resolution of radiolabeled proteins by SDS-PAGE and autoradiography, mass spectrometry identified alpha6-tubulin one of several PKCalpha substrates. Four potential PKC consensus sites of phosphorylation of alpha6-tubulin were identified and mutated to Asp (D) in order to simulate phosphorylation. Motility assays with cells transfected with each mutant identified Ser-165 as the only site in alpha6-tubulin mutants whose pseudo-phosphorylation reproduced the level of motility previously ascribed to PKCalpha. The S165D-alpha6-tubulin was not incorporated into microtubules. Taken together, the results support a model in which phosphorylation of alpha6-tubulin at Ser-165 by PKCalpha leads to cell movement by preventing incorporation of alpha6-tubulin into microtubules.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
