Date of Award

8-7-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Shabb, John B.

Abstract

A fundamental question in the cAMP-dependent protein kinase (PKA) field is how a multi-functional kinase, like PKA, can maintain specificity of a response. Modulating the post-translational levels of the regulatory subunit (RIα) of PKA may be one important means of determining PKA specificity. The Ecdysone-inducible Receptor (EcR)-HEK293 cell line was utilized to examine the hypothesis that an in vivo phosphorylation site at Ser-81 in RIα, located in a putative degradation sequence, may be a signal for protein stability. Pulse-chase experiments determined the half-life of endogenous RIα to be 4.8 hours. A histidine-tagged wild-type or Ser-81-Ala mutant of RIα was stably expressed in the EcR-HEK293 cells. Phosphorylated H6·RI, but not H6·S81A, was identified in cell extracts using a phospho-Ser-81 specific antibody. Western blot analysis determined the half-lives of H6·RI and H6·S81A to be 4.3 and 4.1 hours, respectively. A possible correlation between the phosphorylation state and stability of RIα was also examined in white adipose tissue from mice lacking the RIIβ subunit. The RIIβ knockout model is characterized by a post-translational increase in total RIα levels. Western blot comparisons of knockout and wild-type tissues indicated similar increases in phospho and total RIα levels. Like the signal(s) which determine RIα stability, the mechanistic pathway(s) of RIα degradation are poorly defined. An in vitro rabbit reticulocyte lysate system was used to assess the contribution of phosphorylation at Ser-81 in regulating the ubiquitin/proteasome- or calpain-mediated degradation of RIα. Under the conditions tested the rabbit reticulocyte lysate was unable to degrade RIα. Proteolysis of synthesized RIα was induced by the addition of purified calpain and calcium, though mutation of Ser-81 to Ala in RIα did not influence its susceptibility to degradation by calpain. Finally, the role of phosphorylation at Ser-81 on secondary structure was assessed. Circular dichroism spectroscopy of phosphorylated and non-phosphorylated peptides of RIα detected no structural differences. In summary, the cell culture, adipose tissue, and in vitro systems indicated that phosphorylation at Ser-81 does not play a significant role in determining the stability or secondary structure of RIα Therefore, a function for Ser-81 phosphorylation of RIα remains to be determined.

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