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dc.contributor.advisorStone, Scoten_US
dc.creatorHan, Jiayien_US
dc.date.accessioned2011-06-15T12:36:26Zen_US
dc.date.accessioned2013-01-04T04:38:36Z
dc.date.available2012-06-15T08:00:00Zen_US
dc.date.available2013-01-04T04:38:36Z
dc.date.created2011-06en_US
dc.date.issued2011-06en_US
dc.date.submittedJune 2011en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-06152011-123626en_US
dc.description.abstractTriacylglycerols are the predominant molecules of energy storage in eukaryotes. Triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although the use of molecular tools, including targeted disruption of either DGAT enzyme, has shed light on their metabolic functions, little is known about the mechanisms responsible for regulating DGAT activity. Several lines of evidence from previous studies have suggested that DGAT1, but not DGAT2, is subject to regulation by phosphorylation and that protein kinase A (PKA)-dependent pathways are likely involved. In this study, the role of PKA in regulating DGAT activity and triacylglycerol synthesis during lipolysis was investigated. By using 3T3-L1 adipocytes, in vitro DGAT activity was shown to increase 2 fold during lipolysis. This data suggests that PKA might phosphorylate and activate DGAT1 during lipolysis to promote the recycling/re-esterification of excessive free fatty acids into triacylglycerols before they reach toxic levels within the cell. Additionally, high-performance liquid chromatography – electrospray ionization – mass spectrometry/mass spectrometry was exploited to identify PKA phosphorylation sites of DGAT1, and serine-17, -20 and -25 were identified as potential PKA phosphorylation sites using this methodology. The functional importance of these three potential phosphorylation sites was examined. Mutations of these sites to alanines (to prevent phosphorylation) or aspartates (to mimic phosphorylation) gave rise to enzymes functioning similarly to wild-type DGAT1. These phosphorylation sites appeared to be functionally silent as they were not involved in regulating DGAT1 activity, multimer formation, or enzyme stability. However, PKA phosphorylation at these three sites seemed to play a role in affinity of DGAT1 for its diacylglycerol substrate. These results indicate the existence of other unidentified, functionally active PKA phosphorylation sites or phosphorylation sites of other kinases, which are involved in regulating DGAT1.en_US
dc.language.isoen_USen_US
dc.subjectphosphorylationen_US
dc.subjectprotein kinase Aen_US
dc.subjectmass spectrometryen_US
dc.subjecttriacylglycerolsen_US
dc.subjectacyl-CoA: diacylglycerol acyltransferaseen_US
dc.titleRegulation of acyl-CoA:diacylglycerol acyltransferase-1 by protein phosphorylationen_US
thesis.degree.departmentBiochemistryen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M.Sc.)en_US
dc.type.materialtexten_US
dc.type.genreThesisen_US
dc.contributor.committeeMemberQiu, Xiaoen_US
dc.contributor.committeeMemberKhandelwal, Ramjien_US
dc.contributor.committeeMemberMoore, Stanleyen_US
dc.contributor.committeeMemberPato, Maryen_US


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