Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val6, Ala7]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

The contribution of coagulation factors and fibrinolytic variables to the development of ischaemic arterial disease is still not clearly established. The PRIME study is a prospective cohort study of myocardial infarction in men aged 50-59 years and recruited from three MONICA field centers in France (Lille, Strasbourg and Toulouse) and the center in Northern Ireland (Belfast). Baseline examination included measurement of plasma fibrinogen, factor VII, and PAI-1 activity in over 10,500 participants. We investigated the associations of these haemostatic variables with cardiovascular risk factors, prevalent atherosclerotic disease and geographical area. Fibrinogen level increased with age, smoking, waist-to-hip ratio, LDL-cholesterol, and it decreased with educational level, leisure physical activity, alcohol intake and HDL-cholesterol. Factor VII activity increased with body mass index, waist-to-hip ratio, triglycerides. HDL- and LDL-cholesterol. PAI-1 activity increased with body mass index, waist-to-hip ratio, triglycerides, alcohol intake, smoking, and decreased with leisure physical activity. PAI-1 level was higher in diabetic subjects than in subjects without diabetes. Cardiovascular risk factors explained 8%, 9%, and 26% of the total variance in fibrinogen, factor VII, and PAI-1, respectively. Compared with participants without prevalent cardiovascular disease, those with previous myocardial infarction (n = 280), angina pectoris (n = 230), or peripheral vascular disease (n = 19) had significantly higher levels of fibrinogen. but those with stroke (n = 67) had not. PAI-1 activity showed a similar pattern of association. The odds ratio for cardiovascular disease associated with a rise of a one standard deviation in fibrinogen and PAI-1 was 1.31 (95% confidence interval: 1.20 to 1.42, p <0.001) and 1.38 (95% confidence interval: 1.27 to 1.49, p<0.001), respectively. After adjustment for cardiovascular risk factors, these associations were attenuated but remained highly significant. There was no significant association between factor VII activity and prevalent cardiovascular disease. Fibrinogen level and, to a lesser extent, factor VII and PAI-1 activity were higher in Northern Ireland than France after adjustment for the main cardiovascular risk factors. These geographical variations are consistent with the 2 to 3-fold higher incidence of myocardial infarction in Northern Ireland than France. Our results provide further epidemiological evidence for a possible role of fibrinogen and PAI-1 in the pathogenesis of coronary heart disease.     

Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting

Upon binding of substrates the catalytic subunit (C) of cAMP-dependent protein kinase (cAPK) undergoes significant induced conformational changes that lead to catalysis. For the free apoenzyme equilibrium favors a more open and malleable conformation while the ternary complex of C, MgATP, and a 20-residue inhibitor peptide [PKI (5-24)] adopts a tight and closed conformation [Zheng, J., et al. (1993) Protein Sci. 2, 1559]. It is not clear that binding of either ligand alone is responsible for this conformational switch or whether both are required. In addition, the catalytic subunit binds MgATP and inhibitor peptide synergistically. The structural basis for this synergism is also not defined at present. Using an Fe-EDTA-mediated protein footprinting technique, the conformational changes associated with the binding of MgATP and the heat stable protein kinase inhibitor (PKI) were probed by mapping the solvent-accessible surface and structural dynamics of C. The conformation of the free enzyme was clearly distinguished from the ternary complex. Furthermore, binding of MgATP alone induced extensive conformational changes, both local and global, that include the glycine-rich loop, the linker connecting the small and large lobes, the catalytic loop, the Mg2+ positioning loop, the activation loop, and the F helix. These changes, similar to those seen in the ternary complex, are consistent with a transition from an open to a more closed conformation and likely reflect the motions that are associated with catalysis and product release. In contrast, the footprinting pattern of C.PKI resembled free C, indicating minimal conformational changes. Binding of MgATP, by shifting the equilibrium to a more closed conformation, "primes" the enzyme so that it is poised for the docking of PKI and provides an explanation for synergism between MgATP and PKI.     

Inhibition of the cAMP-dependent protein kinase by synthetic A-helix peptides

The catalytic subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum, PkaC, displays the same properties as its mammalian counterpart, except for being about twice as large in size. Sequence comparisons indicated the presence of a conserved alpha-helix (A-helix) within the N-terminal region of PkaC which could potentially establish close contacts with the catalytic core [Véron, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10618-10622]. We show in this report that a synthetic peptide with the A-helix sequence inhibits PKA activity, whereas unrelated peptides display no inhibitory activity. The inhibition seems competitive with respect to the kemptide substrate rather than due to binding to a secondary site. We further show by amino acid replacements that the last lysine of the A-helix sequence is involved in this specific inhibition. A model is proposed for the possible role of the A-helix.     

Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3' exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3' ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Regulation of ion channels by cAMP-dependent protein kinase and A-kinase anchoring proteins

We cloned a gene for Na+/H+ antiporter from chromosomal DNA of Pseudomonas aeruginosa. Introduction of the gene into host Escherichia coli mutant cells lacking all of the major Na+/H+ antiporters enabled the cells to grow in the presence of 0.2 M NaCl, although the original host cells could not. Membrane vesicles prepared from cells of the transformant possessing the cloned gene showed Na+/H+ antiport activity. As a result of DNA sequencing, we found one open reading frame (nhaP). The deduced amino acid sequence suggests that the Na+/H+ antiporter (NhaP) of P. aeruginosa consists of 424 amino acid residues with molecular mass of 45486 Da, and hydropathy analysis suggested the presence of 12 putative transmembrane domains. We found no bacterial Na+/H+ antiporter which showed significant sequence similarity with the NhaP in the protein sequence database. The NhaP showed partial sequence similarity with animal Na+/H+ exchangers. Thus, the NhaP of P. aeruginosa is unique among bacterial antiporters.     

Modulation of human CACO-2 intestinal epithelial cell phenotype by protein kinase C inhibitors

Protein kinase C (PKC) isoforms are altered in colon tumors and upon exposure of intestinal mucosa to nutrients. We evaluated the effects of the PKC inhibitors staurosporine and calphostin C on human Caco-2 intestinal epithelial proliferation, motility, and differentiation. Motility was quantitated by monolayer expansion and the brush border enzymes dipeptidyl dipeptidase (DPDD) and alkaline phosphatase (AP) by synthetic substrate digestion. Staurosporine (0.03-1.0 ng/ml) and calphostin C (10(-12) M-10(-4) M) dose-dependently inhibited monolayer expansion and AP but stimulated DPDD. Proliferation was also inhibited but the effects of each inhibitor on motility, AP, and DPDD were preserved after mitomycin C proliferative blockade, suggesting that these effects were proliferation-independent. PKC inhibitors independently inhibit motility, AP and proliferation in human intestinal Caco-2 epithelial cells, but selectively stimulate the small intestinal differentiation marker DPDD. PKC may regulate small intestinal epithelial differentiation.     

Regulation of glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase

Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.     

Modulation of AMPA/kainate receptors in cultured murine hippocampal neurones by protein kinase C

1. The patch clamp technique, together with intracellular perfusion of the catalytic fragment of protein kinase C (PKCM), was employed to investigate the role of this enzyme in the intracellular regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors in cultured hippocampal neurones. 2. The responses evoked by near-maximal concentrations of kainate (250 microM) and AMPA (100 microM) were potentiated by the introduction of PKCM, whilst co-application of the inhibitory peptide fragment PKCI(19-36) prevented this action. 3. Modulation of kainate responses by PKCM was dependent upon the concentration of agonist applied. Currents evoked by kainate were potentiated at concentrations above those which caused 50% of the maximal response (EC50) and depressed at lower concentrations. Furthermore, okadaic acid, a specific inhibitor of phosphatases 1 and 2A, had a similar effect upon concentration-response relationships when currents activated by kainate were recorded using the perforated patch technique. 4. In addition, the mean amplitude and/or time constant of decay of miniature excitatory synaptic currents (mediated by AMPA/kainate receptors) was increased by the intracellular injection of PKCM. 5. These observations suggest that the function of postsynaptic excitatory amino acid receptors can be modulated by the activity of PKC as well as by endogenous phosphatases. This regulation may contribute to some forms of synaptic plasticity within the central nervous system.     

Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells.     

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.     

Modulation of regulatory and catalytic subunit levels of cAMP-dependent protein kinase A in anterior pituitary cells in response to direct activation of protein kinases A and C or after GnRH stimulation

We have previously shown that direct activation of protein kinase A (PKA) and protein kinase C (PKC) induced changes in the expression of genes coding for PKA RII beta and C alpha subunit isoforms in cultured anterior pituitary cells, suggesting the possibility of interconnected regulation at this point. To evaluate whether the cell content of PKA protein subunits could be similarly altered, the catalytic (C) and regulatory type I (RI) and type II (RII) subunits were identified by Western blot analysis using specific immunoaffinity-purified antibodies. Activation of PKA by the permeant cyclic adenosine monophosphate (cAMP) analogue 8-Br-cAMP induced a dramatic time- and concentration-dependent decline of C subunit to a residual level that may represent 10-15% of that in untreated cells. The most profound decrease occurred during the first 5 h following treatment with 0.5-2 mM analogue (by 65 +/- 14 and 79 +/- 5%, respectively). Under identical conditions, RII was increased by about 40% at the higher concentrations, while RI increased slightly, but only at low concentrations (below 1 mM 8-Br-cAMP), and then gradually decreased. Exposure of cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) also resulted in decreased levels of the PKA C subunit, however, with a different concentration-dependent profile. In particular, a decline in PKA C was most pronounced (60%) at a low concentration of TPA (10 nM) as compared with the concentrations equal to or above 20 nM (40% decrease). TPA at 10 nM also depressed notably (by 25%) the level of RII subunit, but higher concentrations were essentially ineffective, although a slight and statistically not significant elevation of the cell subunit content was observed as for RI. Simultaneous activation of both PKA and PKC pathways resulted in further depletion of PKA C and an important loss (50%) of RII, a subunit which was enhanced by the activation of either system alone. Finally, gonadotropin-releasing hormone, a neuropeptide that has the potentiality to activate both PKA and PKC signaling in gonadotropes, was able to alter PKA subunit cell content: PKA C was significantly reduced at either a subliminal (0.1 nM) or maximal (10 nM) concentration, whereas RII increased at the low concentration and decreased at the high concentration. In conclusion, these data demonstrate that the pituitary cell contents of RI, RII, and C subunits of PKA are regulated under the activation of PKA itself as well as PKC in a manner that can exhibit further alteration when both systems come simultaneously into play. Changes in the PKA subunit levels in certain cases may correlate with a variation of the mRNAs suggesting multiple control mechanisms, including an alteration of gene expression and changes in subunit degradation, synthesis, and/or turnover. These data, together with those obtained in the presence of gonadotropin-releasing hormone, provide further support for a hormonally induced interplay between PKA and PKC signaling pathways at the crucial level of PKA in the pituitary gland including gonadotropes.     

Effects of lithium on cAMP-dependent protein kinase in rat brain

The essence of the bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) technique is that cells are labelled with the thymidine analogue BrdUrd. They are then allowed to progress through the cell cycle in a BrdUrd-free environment during the postlabelling time period. At a postlabelling time shorter than the length of the S phase (Ts), cells are fixed and prepared for FCM-mediated analysis of BrdUrd and DNA contents. From FCM-derived data, cell cycle kinetic parameters such as labelling index (LI), Ts, and potential doubling time (Tpot) can be calculated. Tpot is believed to be important in the evaluation of tumor aggressiveness and therapy response. Since LI is most commonly used together with Ts to calculate Tpot, it is important that both LI and Ts are independent of the time when cells are sampled. Several formulae to calculate LI and Ts have been presented. In the present paper, we deal with various formulae to calculate LI. These formulae differ in how they take into account unlabelled and BrdUrd-labelled cells in various fractions of the cell cycle. We present a new formula, which takes into consideration cells in the different fractions and thus makes LI theoretically independent of postlabelling time. Our results show that different LI values are obtained when different formulae are used to calculate LI. In addition, we show that the BrdUrd labelling period should be kept as short as possible.     

cAMP-dependent protein kinase in cerebral microvessels in aging and Alzheimer disease

The purpose of this study was to compare the effects of aging and Alzheimer disease (AD) on the important intracellular signaling enzyme cAMP-dependent protein kinase A (PKA) in cerebral microvessels. PKA activity and levels were measured in microvessels isolated from the brains of adult and aged rodents as well as from the cerebral cortices of AD and elderly control patients. The results showed that cerebral microvessels from aged rats have significantly (p < 0.01) higher PKA activity and levels when compared to cerebral microvessels from adult rats. In contrast, no significant difference was found between PKA activity or levels in cerebral microvessels from AD patients when compared to controls. These results indicate that in cerebral microvessels both PKA activity and levels increase with age but are unaffected by AD. The data suggest that protein phosphorylation in brain microvessels may be affected differentially by aging and dementia.     

Cisplatin resistance and regulation of DNA repair in cAMP-dependent protein kinase mutants

Drug resistance in cancer poses a major problem to the success of chemotherapy. Increased resistance to the DNA-damaging chemotherapeutic drug cisplatin may be associated with a variety of factors including decreased drug accumulation, increased intracellular levels of thiols, and increased DNA repair. We have found that mutants of the Chinese hamster ovary (CHO) and the mouse adrenocortical carcinoma Y1 cells harboring a defective regulatory subunit (RI) of the cAMP-dependent protein kinase (PKA) exhibited increased resistance to cisplatin. These mutants are cross-resistant to other DNA-damaging chemotherapeutic agents, including bleomycin and melphalan. In addition, wild-type CHO cells transfected with and overexpressing the yeast phosphodiesterase gene or a dominant mutant Rl alpha subunit gene also displayed similar increased resistance to cisplatin. However, mutants with altered catalytic (C) subunits showed a sensitivity to cisplatin similar to the wild-type cells. Further analysis by gel shift assay using cisplatin-damaged DNA as probes and nuclear extracts derived from the Rl subunit mutants showed increased binding of nuclear factor(s) to the damaged DNA. In addition, a host cell reactivation assay of DNA repair, using a cisplatin-damaged reporter plasmid, detected enhanced capacity for repair of DNA lesions in the PKA mutants. These results suggest that DNA repair may be increased in the PKA mutants. We speculate that functional inactivation of PKA may result in increased DNA repair and the acquisition of resistance to DNA-damaging anticancer drugs in cancer.     

cAMP-dependent protein kinase phosphorylates and activates nuclear Ca2+-ATPase

A Ca2+-pump ATPase, similar to that in the endoplasmic reticulum, has been located on the outer membrane of rat liver nuclei. The effect of cAMP-dependent protein kinase (PKA) on nuclear Ca2+-ATPase (NCA) was studied by using purified rat liver nuclei. Treatment of isolated nuclei with the catalytic unit of PKA resulted in the phosphorylation of a 105-kDa band that was recognized by antibodies specific for sarcoplasmic reticulum Ca2+-ATPase type 2b. Partial purification and immunoblotting confirmed that the 105-kDa protein band phosphorylated by PKA is NCA. The stoichiometry of phosphorylation was 0.76 mol of phosphate incorporated/mol of partially purified enzyme. Measurement of ATP-dependent 45Ca2+ uptake into purified nuclei showed that PKA phosphorylation enhanced the Ca2+-pumping activity of NCA. We show that PKA phosphorylation of Ca2+-ATPase enhances the transport of 10-kDa fluorescent-labeled dextrans across the nuclear envelope. The findings reported in this paper are consistent with the notion that the crosstalk between the cAMP/PKA- and Ca2+-dependent signaling pathways identified at the cytoplasmic level extends to the nucleus. Furthermore, these data support a function for crosstalk in the regulation of calcium-dependent transport across the nuclear envelope.     

Enhancement of the N-methyl-D-aspartate response in spinal dorsal horn neurons by cAMP-dependent protein kinase

Glutamate-gated ion channels mediate excitatory synaptic transmission in the central nervous system and are involved in synaptic plasticity, neuronal development and excitotoxicity (5,24). These ionotropic glutamate receptors were classified according to their preferred agonists as AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), KA (kainate), and NMDA (N-methyl-D-aspartate) receptors [Trends Pharmacol. Sci., 11 (1990) 25-33]. The present study of NMDA receptor channels expressed in acutely isolated spinal dorsal horn (DH) neurons of young rat reveals that they are subject to modulation through the adenylate cyclase cascade. Whole-cell voltage-clamp recording mode was used to examine the effect of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) on the responses of DH neurons to NMDA. Whole-cell current response to NMDA was enhanced by 8 Br-cAMP, a membrane permeant analog of cAMP or by intracellular application of cAMP or catalytic subunit of PKA.     

Mutations in the catalytic subunit of the cAMP-dependent protein kinase interfere with holoenzyme formation without disrupting inhibition by protein kinase inhibitor

Three amino acids were identified in the catalytic (C) subunit of the cyclic AMP-dependent protein kinase that are involved in interaction with regulatory (R) subunit, but not with the specific protein kinase inhibitor, PKI. In a functional assay for gene induction, a C expression vector with serine or arginine substituted for Leu-198 and the double mutant C, His-87-->Gln/Trp-196-->Arg (Orellana, S. A., and McKnight, G. S. (1992) Proc. Natl. Acad. Sci, U.S.A. 89, 4726-4730), retained activity in the presence of an excess of RI or RII. In contrast, cotransfection of a full-length PKI expression vector completely inhibited the activity of both mutant and wild type C subunits. These data suggest that although the substrate/pseudosubstrate sites of R and PKI interact with C at the catalytic site, there is an additional domain on the C subunit that is involved in holoenzyme formation with R subunit and is distinct from sites specifying high affinity PKI binding.