Date of Award

Spring 1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Program/Concentration

Biomedical Sciences

Committee Director

Stephen J. Beebe

Committee Member

Gerald J. Pepe

Committee Member

Howard White

Committee Member

Peter F. Blackmore

Committee Member

Mark S. Elliot

Abstract

The Cα and Cγ of the cAMP-dependent protein kinase (PKA) are two highly homologous (83% amino acid identity), yet functionally distinct isozymes in vitro for substrate and pseudosubstrate specificity, and in intact cells for cell phenotypes (Beebe, 1992).

To determine the molecular mechanisms underlying the distinct functions in cell phenotypes, in vitro experiments were designed to make a detailed comparison of Cγ and Cα for substrate and pseudosubstrate specificity. To this end, Cγ and Cα were expressed m mammalian cells, bacteria and Sf9 insect cells using baculovirus. Abundant expression of active enzyme was cell system specific. Cγ and Cα expressed in Sf9 cells and bacteria, respectively, were purified to homogeneity. Kinetic analysis showed that Cγ and Cα shared primary substrate phosphorylation specificity. Cγ exhibited similar or lower Km values, but lower Vmax values in substrate phosphorylation than Cα. Different isoforms of PKI and R-subunit expressed in bacteria were purified to homogeneity. The kinetic comparison on these pseudosubstrate-inhibition of phosphotransferase activity showed Cγ was insensitive to PKI-inhibition and required the C-terminal residues in the pseudosubstrate site for RIα-inhibition. In vitro data suggested that Cγ had unique substrate and pseudosubstrate specificity. Intact cell experiments were designed to determine the distinct role of Cγ and Cα in the regulation of cAMP-responsive gene expression. To this end, Cγ and Cα were compared in the regulation of CRE (cAMP responsive elements)-reporter gene activity, and in the events which were associated with CRE-reporter gene expression using HEK293 cells and Y1/Kin8 clones permanently transfected with Cγ - and Cα-subunits. In response to cAMP stimulation, both Cγ and Cα migrated to the nucleus, phosphorylated CREB (cAMP responsive element binding protein) and increased the levels of immunoreactive CREB. Phosphorylated CREB in the nuclear extracts of Cγ and Cα clones bound to a CRE element in super gel mobility shift assay. Surprisingly, Cα, but not Cγ induced CRE-reporter gene activity and only Cα clone restored steroidogenesis to cAMP resistant Kin8 mutant. However, Cγ could both positively and negatively modulate Cα-mediated regulation of reporter gene activity. Results from Gal4-luc/Gal4-CREB reporter gene study showed that CRE and CREB dimerization were not necessary for Cγ to modulate the Cα-mediated regulation of CRE-reporter gene activity, suggesting that Cγ and Cα played different roles in the regulation of cAMP-responsive gene activity by an intra-CREB-molecular mechanism.

The presence of two kinetically and functionally distinct C-subunit isoforms provides the potential to fine-tune and/or to diversify cAMP signal transduction downstream of PKA activation, which is mediated by R-subunits and PKI, and/or activation of C-isoforms with different specificity.

Comments

A dIssertation Submitted to the Faculty of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/sjcq-3y57

ISBN

9780591481402

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