Abstract
A mutant form of the type I regulatory subunit (RI) of cAMP-dependent protein kinase has been cloned and sequenced (Clegg, C. H., Correll, L. A., Cadd, G. C., and McKnight, G. S. (1987) J. Biol. Chem. 262, 13111-13119) which contains two point mutations in the site B cAMP-binding site, a Gly to Asp at position this report, the effect of each independent mutation on the rate of dissociation of cAMP from RI, the cAMP-mediated activation of holoenzyme and the inducibility of cAMP-responsive genes has been characterized. Dissociation of cAMP from either recombinant wild type RI or the B1 mutant demonstrated biphasic kinetics, indicating two sites with different affinities for cAMP. Dissociation from the B2 subunit, however, was monophasic and very rapid indicating that site B had been destroyed and that the rate of dissociation from site A was increased. The cAMP activation constants (Ka) of the wild type and B1 holoenzymes were 40 and 188 nM, respectively, and demonstrated positive cooperativity, with Hill coefficients of 1.61 for the wild type and 1.67 for B1. The B2 holoenzyme required much greater concentrations of cAMP, 4.7 microM, for half-maximal activation and did not display positive cooperativity. Constitutive expression in mouse AtT20 pituitary cells of the B1 mutant resulted in only a small shift in the Ka for kinase activation in these cells compared with B2 expression which increased the Ka by more than 100-fold. Transient expression of the B1 subunit in human JEG-3 choriocarcinoma cells inhibited forskolin activation of a cAMP-responsive promoter by 35% whereas similar expression of the B2 RI subunit inhibited the response by 90%. These results suggest that the Gly to Asp mutation at amino acid 324 completely blocks cAMP binding to site B whereas the Arg to His mutation at position 332 causes a more subtle alteration in cAMP binding. Expression of either mutant RI in animal cells results in a dominant repression of cAMP-dependent protein kinase activity and cAMP-dependent protein kinase-mediated processes.
Highlights
A mutant formof the typeI regulatory subunit (RI) function (21, secretion (3), and enzyme activation (4)
Transientexpression of the B1 subunit binding domain of the R subunit which predicts the amino in human JEG-3 choriocarcinoma cells inhibited forskolin activation of a CAMP-responsive promoter by 35%,whereas similar expressionof the B2 RI subunit inhibited the response by 90%.These results suggest that the Gly to Asp mutation at amino acid 324 completely blocksCAMPbinding to sitBe whereas the Arg to His mutation at position 332 causes a more subtle acids involved in a CAMP-bindingpocket
Vectors-Previously, we described the construction of the eucar- Reconstituted CAMP-dependentprotein kinase was dialyzed against yotic expression vectors MT-REV and HL-REV, bothcontaining the TEM andpurified on DEAE-Sephacel
Summary
Inc.) ion exchange column and used to form holoenzyme with bovine Csubunitin the presence of magnesium acetate andATP (17). The previously constructed HL-REV plasmid contained an RI isolation of Stable Clones Expressing Mutant RZ Genes-AtT2O cDNA with an altered site A-coding sequence. Mouse AtT2O pituitary cells (20) weregrown in Dulbecco's obtain the Hill coefficient and half-maximal activation (K.) value modified Eagle's medium plius 10%horse serum (GIBCO) and were with purified holoenzyme, kinase activation curves in the presence of passaged by dissociation in Versene alone. Stable subclones were selected in kinase activity was measured at cAMP concentrations from 2 to 100 media containing 500 pg/ml G-418 (GIBCO) and maintained in 300 p M for the wild type and B1 enzymes, and at cAMP concentrations pg/ml G-418.Cellswere passaged out of G-418 when plated for from 30 to 140 p M for the B2 holoenzyme. The asterisks indicate amino acids identical to the CAP protein thought to bind CAMP.The HinfI restriction site used to separate the B1 andB2 mutations isunderlined Asw considered to he the maximum activity attainable in theassay. The ratio of luciferase to @-galactosidasewas calculated for each sample
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