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Volume 271, Number 26, Issue of June 28, 1996 pp. 15736-15742
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Major Catalytic Subunit Isoforms of cAMP-dependent Protein Kinase Have Distinct Biochemical Properties in Vitro and in Vivo

(Received for publication, February 23, 1996, and in revised form, April 1, 1996)

David M. Gamm , Eric J. Baude ^§ and Michael D. Uhler ^§

From the  Neuroscience Program, the ^§ Department of Biological Chemistry, and the  Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109

Two isoforms of the catalytic subunit of cAMP-dependent protein kinase, C and C1, are known to be widely expressed in mammals. Although much is known about the structure and function of C, few studies have addressed the possibility of a distinct role for the C proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. C1 demonstrated higher K_m values for some peptide substrates than did C, but C1 was insensitive to substrate inhibition, a phenomenon that was observed with C at substrate concentrations above 100 µM. C and C1 displayed distinct IC₅₀ values for the  and  isoforms of the protein kinase inhibitor, protein kinase inhibitor (, , , , , , , , , , , , , , , , , , , ) peptide, and the type II regulatory subunit (RII). Of particular interest, purified type II holoenzyme containing C1 exhibited a 5-fold lower K_a value for cAMP (13 nM) than did type II holoenzyme containing C (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RII₂C1₂ holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RII₂C₂ holoenzyme. These results provide evidence that type II holoenzyme formed with C1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.

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