Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.     

Properties of the regulatory subunit of cAMP-dependent protein kinase type II from human brain

The regulatory subunit type II (RII) of cAMP-dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51-52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One-dimensional peptide mapping of 51-52 kD and 54 kD proteins after digestion with St. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.     

Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways

The intracellular signal transduction pathways utilized by the HIV-1-derived protein, Tat, in the activation of human central nervous system-derived endothelial cells (CNS-ECs) were examined using specific enzymatic assays. Tat induced an increase in interleukin 6 (IL-6) mRNA within 1 hr of treatment. This biological effect of Tat involved activation of both protein kinase C (PK-C) and cAMP-dependent protein kinase (PK-A) in CNS-ECs. Tat at 10 ng/ml induced a sharp, transient increase in membrane PK-C activity within 30 sec of incubation, and reached maximum levels at 2 min, declining to control values within 10 min. Tat also induced a sharp increase in intracellular cAMP levels and PK-A activity in these cells, with the PK-A activity reaching a maximum at 10 min and slowly declining to control values in 4 hr of incubation. Activation of PK-A was dependent on a Tat-induced increase in membrane PK-C activity as demonstrated by calphostin C (a PK-C inhibitor) abolishing this effect. Incubation of cells with the cyclooxygenase inhibitor indomethacin did not affect Tat-induced activation of PK-A, indicating that prostacyclins are not involved in this process. Tat-induced increase in IL-6 mRNA was abolished in the presence on PK-A inhibitor H-89, demonstrating that activation of PK-A is necessary and sufficient for the increase in IL-6 production by these cells. Both the Tat-induced increase in intracellular cAMP and IL-6 mRNA levels in CNS-ECs may play a role in altering the blood-brain barrier and thereby inducing pathology often observed in AIDS dementia.     

Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA alpha-isoforms RIalpha, RIIalpha, and Calpha and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.     

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.     

Nitric oxide regulates cyclic GMP-dependent protein kinase phosphorylation in rat brain

Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.     

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.     

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.     

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.     

Participation of ADP dissociation in the rate-determining step in cAMP-dependent protein kinase

Pre-steady-state kinetic analyses of the catalytic subunit of cAMP-dependent protein kinase showed that the rate constant for phosphoryl transfer is fast and either the release of one or both of the products or a conformational change controls turnover [Grant, B., & Adams, J. A. (1996) Biochemistry 35, 2022-2029]. To determine which step or steps control turnover in the wild-type enzyme, we used a catalytic trapping technique to measure directly the dissociation rate constant for ADP. The phosphorylation of two peptide substrates, LRRASLG and GRTGRRNSI, was monitored using a rapid quench flow technique under conditions where saturating concentrations of ADP were preequilibrated with the enzyme before excess ATP and one of the substrates were added to trap the free enzyme and to start the phosphorylation reaction. Under ADP preequilibration conditions, no 'burst' phase was observed, and although the rate of linear, steady-state turnover was unaffected, the net production of phosphopeptide lagged behind the non-preequilibrated control. This phenomenon occurs due to the slow release of the product, and kinetic modeling suggests that this effect can be explained if the dissociation rate constant for ADP is 24 s-1 and solely limits turnover (kcat = 23 s-1) for the phosphorylation of LRRASLG. Using GRTGRRNSI, the dissociation rate constant for ADP is 35 s-1 and limits turnover (kcat = 29 s-1) if the reaction is initiated by the addition of enzyme. Under preequilibration conditions with either ATP or GRTGRRNSI, turnover is approximately 50% lower, suggesting that ADP release may partially control this parameter. This preequilibration effect can be explained by slowly interconverting enzyme forms with specific peptide-induced turnover properties. These studies indicate that ADP release is an essential rate-limiting component for turnover but also suggests that other factors contribute subtly when the structure of the substrate is altered.     

Effects of chronic lithium and electroconvulsive stimuli on cholecystokinin mRNA expression in the rat brain

This study compares the effect of lithium (Li+) and electroconvulsive stimuli (ECS), two treatments commonly used in the treatment of affective disorders, on CCK mRNA expression in the rat brain. Two groups of rats receiving either 4 week Li+ or vehicle food supplementation and two groups receiving 6 ECS or 6 sham ECS during 2 weeks were studied. A significant decrease in CCK mRNA levels was seen in the caudate putamen both after Li+ as compared to vehicle and ECS as compared to sham ECS, 27 and 25%, respectively. A small (10%), yet significant, decrease was also seen in the inner entorhinal cortex after Li+. The results indicate that both Li+ and ECS inhibit CCK synthesis in the caudate putamen and are consistent with other findings of presumed decreased dopaminergic action in this part of the brain following these treatments.     

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.     

Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.     

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.     

N-myristylation of the catalytic subunit of cAMP-dependent protein kinase conveys structural stability

Coexpression of the yeast N-myristyltransferase with the murine catalytic subunit of cAMP-dependent protein kinase in prokaryotic cells results in the N-myristylation of the recombinant catalytic subunit. The acylated recombinant catalytic subunit was purified following in vitro holoenzyme formation with a mutant form of the regulatory subunit and compared to the non-myristylated recombinant enzyme and to the mammalian porcine enzyme. All three enzymes are very similar in terms of their kinetic properties and their capacity to reassociate in vitro with the regulatory subunit to form holoenzyme. In contrast, the myristylated recombinant catalytic subunit is significantly more stable to thermal denaturation than the non-myristylated enzyme. Its thermal stability is now comparable to the mammalian enzyme. All three catalytic subunits are significantly more stable to thermal denaturation when they are part of the holoenzyme complex. Each shows an increase in T1/2 of 10 degrees C. This study demonstrates that one function for the myristic acid at the NH2 terminus of the catalytic subunit is to provide structural stability.     

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.     

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.     

Effects of microgravity and bone morphogenetic protein II on GFAP in rat brain

Enzyme activities and protein levels of several protein and lipid kinases were measured in postmortem tissue from patients who died with amyotrophic lateral sclerosis (ALS) as well as from control subjects. Patients who died with ALS had increased activities and protein levels of phosphatidylinositol 3-kinase (PI 3-K) in particulate fractions of spinal cord tissue compared with control subjects. The PI 3-K activity increased with PI 3-K protein level, indicating no change in specific PI 3-K activity in ALS. No differences in PI 3-K activities were found in cytosolic fractions of spinal cord, or in motor and visual cortices, from ALS patients compared with those from controls. PI 3-K activities and protein levels were unchanged in brain tissue from patients who died with Alzheimer's disease compared with those from controls. PI 3-K is a lipid kinase that is important for cell survival and is activated in response to many growth factors. Increased PI 3-K activities in particulate fractions of spinal cord from ALS patients may be related to the increase of PI 3-K protein levels found in this tissue. The protein kinases Erk2, protein kinase B (PKB), and p70 ribosomal S6 kinase (S6K) showed no differences in activities in spinal cord tissue between ALS patients and controls. However, the amounts of PKB and S6K protein were significantly higher in ALS patients, whereas Erk2 protein amount was unchanged compared with controls. Protein kinase C activity was increased in spinal cord tissue from ALS patients, which is consistent with our previous report. The increased activity of PI 3-K in spinal cord tissue from patients with ALS implicates the involvement or activation of PI 3-K in ALS, as either a cause or a consequence of the neuron loss. The lack of up-regulation in the activities of PKB and S6K in ALS tissue supports an impairment in signal transduction cascades mediated by PI 3-K in this neurodegenerative disease.     

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.