Date of Award

5-5-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

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Abstract

The mechanism by which the type la regulatory subunit (RIa) of cAMP-dependent protein kinase (PKAI) is localized to cell membranes is unknown, however, RIa may be localized through hydrophobic interaction with membrane lipids. To determine if post-translational lipid modification of RIa is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane and cytosolic forms of RI after digestion with trypsin or AspN protease. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIa gene product Mass spectral analysis also indicated that membrane-associated RIa had a higher extent of disulfide bond formation in the amino-terminal dimerization domain than did cytosolic RIa. A single RIa phosphorylation site was identified at Ser-81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIa. Because both R subunit preparations were 30-40% phosphorylated, this post-translational modification could not be responsible for the compartmentation of the majority of RIa. Lack of detectable structural differences between membrane-associated and cytosolic RIa strongly suggest an interaction between RIa and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIa membrane association. Although RIa lacked lipid modifications, determination o f the phosphorylation site resulted in a new area of interest The role of Ser-81 phosphorylation of RIa is unknown. It has been reported that the hearts of spontaneously hypertensive rats have reduced levels of PBCAI relative to normotensive rats. It was hypothesized that if phosphorylation modulated the half-life of RIa, then there may be a difference in phosphorylation state o f RIa from the hearts. Western blot analysis using antibodies against RIa, and antibodies directed specifically against phospho-Ser-81, were used to show relative changes in total RIa and phosphorylated RIa levels between normotensive and hypertensive rats. A survey of eight rat tissues showed ubiquitous RIa phosphorylation. Multivariate analysis of variance indicated that total and phosphorylated RIa levels in hypertensive rats were different from those in normotensive rats. The tissues that contributed most to the difference in RIa levels were heart, kidney, and testis

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