Cγ- and Cα-Subunits Provide the Specificity for cAMP/PKA Function
type: CreativeWork
description: 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 e…
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 e…
author: Zhang, Wei Qing
keywords: Catalytic subunit gamma, Protein kinase A, Cell Biology, Molecular Biology
date published: 1997-04-01T08:00:00Z
date modified: 2026-07-24T03:35:30Z
provider: Old Dominion University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Old Dominion University