The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo.

J Biol Chem 1996 Jun;271(26):15736-42

Department of Biological Chemistry, and the Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.

Two isoforms of the catalytic subunit of cAMP-dependent protein kinase, Calpha and Cbeta1, are known to be widely expressed in mammals. Although much is known about the structure and function of Calpha, few studies have addressed the possibility of a distinct role for the Cbeta 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. Cbeta1 demonstrated higher Km values for some peptide substrates than did Calpha, but Cbeta1 was insensitive to substrate inhibition, a phenomenon that was observed with Calpha at substrate concentrations above 100 microM. Calpha and Cbeta1 displayed distinct IC50 values for the alpha and beta isoforms of the protein kinase inhibitor, protein kinase inhibitorpeptide, and the type IIalpha regulatory subunit (RIIalpha). Of particular interest, purified type II holoenzyme containing Cbeta1 exhibited a 5-fold lower Ka value for cAMP (13 nM) than did type II holoenzyme containing Calpha (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 RIIalpha2Cbeta12 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 RIIalpha2Calpha2 holoenzyme. These results provide evidence that type II holoenzyme formed with Cbeta1 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.

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.271.26.15736DOI Listing
June 1996

Publication Analysis

Top Keywords

protein kinase
16
calpha cbeta1
12
type holoenzyme
12
lower concentrations
8
cells expressing
8
camp-dependent protein
8
5-fold lower
8
biochemical properties
8
catalytic subunit
8
cos-1 cells
8
holoenzyme
6
calpha
6
cbeta1
6
values alpha
4
ic50 values
4
distinct ic50
4
concentrations forskolin
4
forskolin cos-1
4
isoforms protein
4
kinase inhibitorpeptide
4

References

(Supplied by CrossRef)

Wiemann et al.
J. Biol. Chem. 1991

Uhler et al.
J. Biol. Chem. 1986

Adavani et al.
Eur. J. Biochem. 1987

Showers et al.
J. Biol. Chem. 1986

Uhler et al.
J. Biol. Chem. 1987

Similar Publications