Abstract
The complex of the subunits (RIalpha, Calpha) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K(d) = 0.25 microm) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RIalpha or RIIalpha was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RIalpha or RIIalpha showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.
Highlights
The cAMP-dependent protein kinase1 differs from other kinases in having the catalytic site and the autoinhibitory site on two different subunits
We used the cAMP-responsive element (CRE)-luciferase reporter gene to probe for dissociation of cAMP-dependent protein kinase I (cA-PKI) and cA-PKII in intact cells
The effect of expressed exogenous RI and C subunits could be mutually titrated in cells not stimulated with cAMP agonists (Fig. 1, B and C), confirming that the CRE-Luc expression system responded to C␣ subunit and that the C␣ effect could be blocked by R subunit
Summary
Fluorescein 5-isothiocyanate (FITC) and tetramethylrhodamine-5isothiocyanate (TRITC) were from Molecular Probes, Eugene, OR. The C-FITC and the biotinylated C subunit had the same molar activity (18 sϪ1) as unmodified C, and similar efficiency in releasing [3H]cAMP bound to RI␣. Assay of Protein Kinase Activity and of R Subunit [3H]cAMP Binding Activity—Kinase incubations were in buffer A with [␥-32P]ATP. Estimation of the Level of R and C Subunits of cA-PK, and of C Subunit Occupation by Substrate in Intact cAMP-stimulated Cells—A thorough study [24] showed equimolar expression of the R (RI ϩ RII) and C subunits of cA-PK in mammalian tissue, averaging 0.3 pmol/mg tissue, wet weight This translates to an average intracellular concentration of subunits of about 0.5 M, assuming the extracellular space to occupy 15% of the tissue, and the subunits to distribute in 70% of the intracellular space. The phosphorylation was about 8 times slower in the intact cell, suggesting that one-eighth of the C subunit pool was accessible for PAH phosphorylation, and that at most seven-eights of it (85–90%) was occupied by other substrates
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