Codominant expression of a mutation affecting the cAMP-dependent protein kinase catalytic subunit in somatic cell hybrids of LLC-PK1 cells

The LLC-PK1 mutant cell lines FIB4 and FIB6 are affected in the catalytic (C) subunit of cAMP-dependent protein kinase (cAMP-PK) such that they possess less than 10% parental activity. However, by Western blot analysis they were shown to possess normal levels of C subunit protein. Somatic cell hybrids were derived between mutant and LLC-PK1 cells, and examined for complementation of the cAMP-PK lesion. Codominant expression of mutant and normal alleles was observed, in that somatic cell hybrids between FIB4 and LLC-PK1, and between FIB6 and LLC-PK1 cells, exhibited cAMP-PK activity 60-75% that of LLC-PK1 cells, intermediate between mutant and normal parental cell lines. The cAMP-PK of the FIB6 x LLC-PK1 and FIB4 x LLC-PK1 hybrids was examined by ion exchange chromatography. In contrast to the FIB6 and FIB4 mutants which lack an active Type I cAMP-PK, the hybrids retained levels of active Type I cAMP-PK greater than 30% that of LLC-PK1, concomitant with the retention of catalytic activity. It was concluded that the loss of Type I kinase in the FIB6 and FIB4 mutants is most likely a consequence of the lesion in the cAMP-PK C subunit. All somatic cell hybrids examined showed levels of cAMP-PK C subunit (as determined by Western blot analysis), and in vivo regulation of cAMP-PK activation (in response to hormonal or nonreceptor-mediated stimulation of adenylate cyclase), completely comparable to those of the parental LLC-PK1 cells. Hence, no aberrant regulation of either cAMP-PK subunit levels or cAMP-PK activities was evident in the somatic cells hybrids. All data were consistent with the hypothesis that FIB4 and FIB6 contain a structural mutation affecting the cAMP-PK catalytic subunit that is expressed phenotypically in the presence of the normal allele.     

Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.     

LLC-PK1 cell mutants in cAMP metabolism respond normally to phorbol esters

Mutants of the pig kidney cell line, LLC-PK1, affected in cAMP metabolism, were examined for cAMP-dependent protein kinase (cAMP-PK) activity and for cAMP-mediated induction of urokinase-type plasminogen activator (uPA). The FIB4 and FIB6 mutant cell lines possessed about 10% parental levels of cAMP-PK activity and concomitantly reduced uPA production (10-20% parental) in response to calcitonin, forskolin and 8-bromo cAMP. The FIB1, FIB2 and FIB5 mutant cell lines had about 70% parental levels of cAMP-PK and the synthesis of uPA was 40-60% parental. Thus, cAMP-mediated induction of uPA showed a dependence on the absolute levels of cAMP-PK. However, uPA synthesis in response to phorbol-12-myristate-13-acetate by all of the mutants was similar to parental, which indicates that enzyme induction mediated by phorbol esters does not involve cAMP or cAMP-PK.     

Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells.

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The 'receptorless' mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.     

cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells.

The relationship between activation of the cAMP-dependent protein kinase (cAMP-PK) and ligand binding and internalization by the vasopressin renal (V2-type) receptor of LLC-PK1 renal epithelial cells was examined. Upon cAMP-PK activation through 1 h treatment with the cAMP analogue 8-bromo-cAMP (BrcA), a marked reduction in V2-receptor steady state number and internalization in LLC-PK1 cells was effected. In cells treated for 17 h with BrcA and hence down-regulated for cAMP-PK, the V2-receptor number was normal but internalization was markedly reduced. Cells of the LLC-PK1 mutant FIB4, which possesses about 10% parental cAMP-PK catalytic subunit activity, exhibited lower V2-receptor steady state number and internalization in comparison to untreated LLC-PK1 cells. A negative correlation was thus evident between cAMP-PK activation and V2-receptor number, and internalization. Phosphorylation by cAMP-PK may effect ligand-independent removal of receptor from the plasma membrane.     

In situ reassociation of the regulatory and catalytic subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase isoenzymes in AtT20 cells

Dissociation and reassociation of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases I and II were studied in intact AtT20 cells. Cells were stimulated with 50 microM forskolin to raise intracellular cAMP levels and induce complete dissociation of R and C subunits. After the removal of forskolin from the incubation medium cAMP levels rapidly declined to basal levels. Reassociation of R and C subunits was monitored by immunoprecipitation of cAMP-dependent protein kinase activity using anti-R immunoglobulins. The time course for reassociation of R and C subunits paralleled the loss of cellular cAMP. Total cAMP-dependent protein kinase activity and the ratio of protein kinase I to protein kinase II seen 30 min after the removal of forskolin was the same as in control cells. Similar results were seen using crude AtT20 cell extracts treated with exogenous cAMP and Mg2+. Our data showed that after removal of a stimulus from AtT20 cells inactivation of both cAMP-dependent protein kinase isoenzymes occurred by the rapid reassociation of R and C subunits to form holoenzyme. Our studies also showed that half of the type I regulatory subunit (RI) present in control cells contained bound cAMP. This represented approximately 30% of the cellular cAMP in nonstimulated cells. The cAMP bound to RI was resistant to hydrolysis by cyclic nucleotide phosphodiesterase but was dissociated from RI in the presence of excess purified bovine heart C. The RI subunits devoid of C may function to sequester cAMP and, thereby, prevent the activation of cAMP-dependent protein kinase activity in nonstimulated AtT20 cells.     

Functional characterization of cAMP-binding mutations in type I protein kinase

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.     

Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation

The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.     

Reduced levels of cardiac cAMP-dependent protein kinase in spontaneously hypertensive rat

Cardiac cAMP-dependent protein kinases were compared between the spontaneously hypertensive rat and the age-matched normotensive Wistar-Kyoto rat by DEAE-cellulose chromatography, photoaffinity labeling with 8-N3[32P]cAMP, and Western blots using the antiregulatory and 125I-anticatalytic subunit antibodies. DEAE-cellulose chromatography revealed that the ratio of type I to type II cAMP-dependent protein kinase was 3:1 in the cytoplasmic soluble proteins from the heart of normotensive rat. In contrast, the ratio of type I to type II was 1:1 in the heart of hypertensive rat. Type I protein kinase was reduced by 3-fold in hypertensive rat compared to normotensive rat. The levels of type II protein kinase were similar in both normotensive and hypertensive rats. The ratio of regulatory subunits of type I (RI) to type II (RII) cAMP-dependent protein kinase was 2.5 in the soluble proteins from the heart of normotensive rat compared to a ratio of 0.62 for hypertensive rat. RI was reduced by 4-fold in hypertensive rat compared to normotensive rat. The decrease in RI from hypertensive rat was also demonstrated by photoaffinity labeling with 8-N3[32P] cAMP. Western blot analysis of the catalytic subunit revealed a 2-fold decrease in catalytic subunit (C) in the soluble proteins from the hypertensive rat compared to normotensive rat. These results show that the reduced level of activity of cardiac type I protein kinase in hypertensive rat was the result of a decrease in both the RI and C subunits, thus reducing the number of type I cAMP-dependent protein kinase holoenzyme molecules. Comparison of type I protein kinase from "prehypertensive" and "hypertensive" stages of hypertensive rat indicated that the type I protein kinase was reduced by 3-fold before an increase in the blood pressure was detectable. Cardiac type I protein kinase is predominantly associated with the cytoplasmic proteins in both the normotensive and hypertensive rats. The levels of RI, RII, and C associated with the membrane-solubilized proteins were not affected in the hypertensive rat. The levels of RII were similar in the brain tissue of normotensive and hypertensive rats, suggesting that the decrease in type I protein kinase is specific in hypertensive rat. In conclusion, a decrease in cardiac type I cAMP-dependent protein kinase may affect the degree of phosphorylation of cardiac regulatory proteins, thus impairing normal cardiac physiology in hypertensive rat.     

Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line.

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.     

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Molecular basis for isoform-specific autoregulation of protein kinase A

Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P(0)-site and RII subunits being a substrate. Under physiological conditions, RII does not inhibit PrKX, the human X chromosome encoded PKA catalytic (C) subunit. Using a live cell Bioluminescence Resonance Energy Transfer (BRET) assay, Surface Plasmon Resonance (SPR) and kinase activity assays, we identified the P(0)-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme formation only takes place with an alanine at position P(0), whereas RI subunits containing serine, phosphoserine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P(0) from serine to alanine. In contrast, PKA-Calpha forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type II or mutant PKA type I holoenzymes (P(0): Ser or Asp) are able to dissociate fully upon maximally elevated intracellular cAMP. The data are of particular significance for understanding PKA isoform-specific activation patterns in living cells.     

Coelution of the type II holoenzyme form of cAMP-dependent protein kinase with regulatory subunits of the type I form of cAMP-dependent protein kinase

The types and subunit composition of cAMP-dependent protein kinases in soluble rat ovarian extracts were investigated. Results demonstrated that three peaks of cAMP-dependent kinase activity could be resolved using DEAE-cellulose chromatography. Based on the sedimentation of cAMP-dependent protein kinase and regulatory subunits using sucrose density gradient centrifugation, identification of 8-N3[32P]cAMP labeled RI and RII in DEAE-cellulose column and sucrose gradient fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Scatchard analysis of the cAMP-stimulated activation of the eluted peaks of kinase activity, the following conclusions were drawn regarding the composition of the three peaks of cAMP-dependent protein kinase activity: peak 1, eluting with less than or equal to 0.05 M potassium phosphate, consisted of the type I form of cAMP-dependent protein kinase; peak 2, eluting with 0.065-0.11 M potassium phosphate, consisted of free RI and a type II tetrameric holoenzyme; peak 3, eluting with 0.125 M potassium phosphate, consisted of an apparent RIIC trimer, followed by the elution with 0.15 M potassium phosphate of free RII. The regulatory subunits were confirmed as authentic RI and RII based upon their molecular weights and autophosphorylation characteristics. The more basic elution of the type II holoenzyme with free RI was not attributable to the ionic properties of the regulatory subunits, based upon the isoelectric points of photolabeled RI and RII and upon the elution location from DEAE-cellulose of RI and RII on dissociation from their respective holoenzymes by cAMP. This is the first report of a type II holoenzyme eluting in low salt fractions with free RI, and of the presence of an apparent RIIC trimer in a soluble tissue extract.     

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.     

Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase

Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.     

The consequences of introducing an autophosphorylation site into the type I regulatory subunit of cAMP-dependent protein kinase

The type I and type II regulatory subunits of cAMP-dependent protein kinase can be distinguished by autophosphorylation. The type II regulatory subunits have an autophosphorylation site at a proteolytically sensitive hinge region, while the type I regulatory subunits have a pseudophosphorylation site. Only holoenzyme formed with type I regulatory subunits has a high affinity binding site for MgATP. In order to determine the functional consequences of regulatory subunit phosphorylation on interaction with the catalytic subunit, an autophosphorylation site was introduced into the type I regulatory subunit using recombinant DNA techniques. When Ala97 at the hinge region of the type I regulatory subunit was replaced with Ser, the regulatory subunit became a good substrate for the catalytic subunit. Stoichiometric phosphorylation occurred exclusively at Ser97. Radioactivity was incorporated primarily into the recombinant regulatory subunit when catalytic subunit and [gamma-32P]ATP were added to the total bacterial extract. Phosphorylation of the mutant regulatory subunit also occurred readily following polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose. Phosphorylation occurred as an intramolecular event in the absence of cAMP indicating that the hinge region of the regulatory subunit occupies the substrate recognition site of the catalytic subunit in the holoenzyme complex. Holoenzyme formed with both the wild type and mutant regulatory subunits was susceptible to dissociation in the presence of high salt; however, only the native holoenzyme was stabilized by MgATP. In contrast to the wild type holoenzyme, the affinity of the mutant holoenzyme for cAMP was not reduced in the presence of MgATP. Holoenzyme formation also was not facilitated by MgATP.     

Inhibitory effect of the regulatory subunit of type I cAMP-dependent protein kinase on phosphoprotein phosphatase

The regulatory subunit of type I cAMP-dependent protein kinase (RI) from rabbit skeletal muscle inhibited the activity of a low molecular weight phosphoprotein phosphatase. The inhibition was concentration and time dependent. A maximum inhibition, about 70%, was observed at 2 microM of RI with an apparent Ki of 0.8 microM. Inhibition was associated with a decrease in Vmax with no change in Km for substrate, phosphorylase a. On the other hand, cAMP-dependent protein kinase holoenzyme or its catalytic subunit was without any effect. The inhibition of phosphoprotein phosphatase by RI may be of physiological significance since the dissociation of cAMP-dependent protein kinase by cAMP would result in a simultaneous increase in the phosphorylation and decrease in the dephosphorylation rates of target proteins.     

Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists.

We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.     

Involvement of cAMP and cAMP-dependent protein kinase in the initiation of DNA synthesis by rat liver cells

Addition of calcium to calcium-deprived cultures of T51B rat liver cells caused brief bursts of cAMP production and cAMP-dependent protein kinase activity which were followed almost immediately by a stimulation of DNA synthesis. PKInh, a specific polypeptide inhibitor of the catalytic subunits of cAMP-dependent protein kinases, inhibited the DNA-synthetic response to calcium addition without stopping the preceding cAMP surge. Addition of cAMP to the calciumdeprived cultures increased protein kinase activity and stimulated DNA synthesis, both of which were inhibited by PKInh. DNA synthesis in these cultures was not stimulated by adding type I cAMP-dependent protein kinase holoenzyme to the calcium-deficient medium, but it was stimulated by type II cAMP-dependent protein kinase holoenzyme or the catalytic subunit from either type I or type II holoenzyme. The stimulatory actions of the type II holoenzyme or the catalytic subunits were inhibited by PKInh. Thus, a burst of cAMP-dependent protein kinase activity was ultimately responsible for the stimulation of DNA synthesis in calcium-deprived T51B cells by calcium or cAMP and it might also be involved in the events leading to initiation of DNA synthesis in many, if not all, normally cycling cells.     

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.