Protein kinases in amphibian ectoderm induced for neural differentiation

Summary Ectoderm explants from early gastrula stages of Xenopus laevis were induced with a neutralizing factor. The factor was isolated from Xenopus gastrulae and partially purified by chromatography on DEAE cellulose. The ectoderm was cultured for different periods of time and then homogenized. Protein kinase activity was determined in the homogenates from induced and control explants with histone H 1 or C-terminal peptide derived from histone H 1 as substrates. The C-terminal peptide is a more specific substrate for protein kinase C, whereas histoneH 1 is a substrate for cAMP/cGMP-dependent protein kinases as well protein kinase C. With both substrates the enzyme activity increases after induction. With the C-terminal peptide as the substrate the protein kinase activity is lower, but its relative increase after induction higher. This suggests that besides cAMP/cGMP dependent protein kinases protein kinase C or related enzymes are involved in the neural induction and differentiation processes. This corresponds to previous experiments which have shown that treatment of ectoderm with phorbol myristate acetate, an activator of protein kinase C and protein kinase C related enzymes, initiates neural differentiation. Endogeneous substrates, which are more intensively phosphorylated after induction are proteins with apparent molecular weights 21 kDa and 31 kDa. Addition of protein kinase C to the induced and control homogenates abolishes the difference in the phosphorylation rate of these proteins.     

A unique cyclic nucleotide-dependent protein kinase

During the course of studying the soluble cyclic nucleotide-dependent protein kinases of a developing insect, three different enzymes were isolated. Two of these were found to be cAMP-dependent enzymes eluting from DEAE-cellulose in a manner identical with protein kinases I and II found in vertebrate muscle. The third enzyme appears to be unique. It has high affinity for either cAMP or cGMP (KA of 43 nM and 25 nM, respectively), the only cyclic nucleotide-dependent kinase described, to have this property. The enzyme has lower affinity for cIMP and cCMP (KA of 160 nM and 340 nM, respectively). Binding to cyclic nucleotide does not alter enzyme size. The KM for ATP is 86 microM, and among several types of histones tried, the slightly lysine-rich subgroup f2a was the best phosphate acceptor. Maximum activity was obtained with 1 mM Mg2+ while Mn2+ was completely ineffective. This new enzyme was purified to homogeneity on a cAMP affinity column as judged by two-dimensional electrophoresis. On the basis of molecular sieving and sodium dodecyl sulfate electrophoresis we have reached the preliminary conclusion that the native enzyme is a dimer of identical subunits with a molecular weight of 180,000. If the mammalian cAMP and cGMP enzymes are indeed homologous proteins, perhaps we have in this new kinase a species that represents a common ancestral protein.     

An unusual cyclic AMP-dependent protein kinase from nematodes

The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode $\text{Aphelenchus}$$\text{avenae}$. One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the $\text{cAMP}\text{cGMP}$ kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation.     

Isolation and preliminary characterization of a casein kinase from cauliflower nuclei

A casein-type protein kinase has been isolated from cauliflower (Brassica cauliflora Gars.) nuclei and purified to a specific activity of 23,000 units/milligram of protein (1 unit is defined as the transfer of 1 picomole of ³²Pi from γ-[³²P]ATP to substrate per minute at 28 C). The enzyme has a molecular weight of approximately 39,000 as judged by sucrose density gradient sedimentation. The casein kinase requires ATP as the phosphate donor and will phosphorylate casein and phosvitin, but not histones. The enzyme activity is not affected by cAMP or cGMP. The casein kinase appears to be analogous to casein kinases described in other plant and animal systems.     

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

A protein phosphorylation-based assay for screening and monitoring of drugs modulating cyclic nucleotide pathways

Cyclic nucleotide regulation is an important target for drug development, particularly for treatment and prophylaxis of cardiovascular diseases. Determination of cyclic nucleotide levels for screening and monitoring of cyclic nucleotide modulating drug action is necessary, yet the techniques available are cumbersome and not sufficiently accurate. Here we present an approach based on the detection of cyclic nucleotide-dependent protein phosphorylation. By use of a common substrate of cyclic nucleotide-dependent protein kinases, the protein vasodilator-stimulated phosphoprotein (VASP) featuring two phosphorylation sites specifically phosphorylated by these kinases, an assay was developed for the monitoring of intracellular cyclic nucleotide levels. The assay was tested with human platelets ex vivo treated with stimulants of nucleotide cyclases, kinases, and phosphodiesterase inhibitors. Phosphorylation of the protein VASP correlates with intracellular cyclic nucleotide concentration (R² > 0.90 for cGMP and cAMP); however, VASP phosphorylation is more sensitive to elevated cyclic nucleotide levels and significantly more stable over time. Quantification of VASP phosphorylation offers a reliable and robust tool for fast and easy monitoring of cyclic nucleotide levels and is also applicable to unprocessed biological matrices. Owing to these properties, VASP is a promising biomarker for screening and monitoring of cyclic nucleotide modulating drugs.     

CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES FROM SILKWORM EGGS: OCCURRENCE OF TWO CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES, CHANGES IN THEIR ACTIVITIES AND LEVELS OF CYCLIC NUCLEOTIDES DURING DEVELOPMENT OF THE EGGS

Two cyclic nucleotide-dependent protein kinases, designated as protein kinase-I and -II, were obtained from the eggs of the silkworm, Bombyx mori . Protein kinase-I is highly dependent on cGMP, whereas protein kinase-II is dependent on cAMP. In developing non-diapause eggs, the level of cyclic nucleotide-dependent protein kinase activity is quite high but that in the diapause eggs is not. The developmental changes in the two protein kinases and the level of cyclic nucleotides were also studied during the development of the eggs.     

Improved two-step method for the assay of adenylate and guanylate cyclase.

A two-step assay for adenylate and guanylate cyclase is described utilizing α-³²P-labeled ATP or GTP as substrate and involving purification of the resulting ³²P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 mm HCl for cGMP and 10 mm HClO₄ for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the ³²P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.     

Phosphofructokinase from the anterior byssus retractor muscle of Mytilvs edulis: Modification of the enzyme in anoxia and by endogenous protein kinases

• 1.1. Anoxia exposure resulted in a stable modification of the kinetic properties of 6-phosphofructo-1-kinase (PFK) from the anterior byssus retractor muscle (ABRM) of the sea mussel Mytilus edulis L.
• 2.2. Compared to the aerobic enzyme, the anoxic form of PFK. showed a reduced affinity for both substrates, fructose-6-phosphate (F6P) and ATP, and an increased sensitivity to inhibition by phosphoenolpyruvate.
• 3.3. To analyze the involvement of protein kinases in the modification of PFK, extracts from aerobic or anoxic muscle were incubated with ATP and Mg²⁺ plus protein kinase second messengers cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP) or Ca²⁺ plus phorbol 12-myristate 13-acetate (PMA).
• 4.4. Both forms of the enzyme responded to the presence of cAMP with a strong increase in affinity for F6P.
• 5.5. In response to cGMP affinity of the aerobic enzyme for F6P decreased whereas that of the anoxic enzyme form was not affected (at 0.5 mM ATP) or increased (at 3 mM ATP).
• 6.6. Incubation with Ca²⁺ + PMA had only a limited effect on PFK kinetics but appeared to enhance the response to cGMP when the three compounds were given together.
• 7.7. Treatment of PFK-aerobic with alkaline phosphatase resulted in a strong decrease in enzyme activity and affinity for F6P; subsequent treatment with cAMP reversed the effect on S_0.5 F6P.
• 8.8. The data indicate that PFK activity is altered during the aerobic-anaerobic transition by a change in the phosphorylation state of the enzyme and that cAMP and cGMP act oppositely to regulate PFK activity, and thereby alter glycolytic rate, during this transition.

     

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca²⁺-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca²⁺-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca²⁺ and CDR. In subcellular fractionation experiments both the CDR and the Ca²⁺-dependent phosphodiesterase were predominantly located in the 100,000g supernatant fraction.The apparent K_m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μm, respectively, when CDR was not rate limiting. Minor increases in the apparent K_m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μm Ca²⁺ for the hydrolysis of 25 μm cGMP and by 8 μm Ca²⁺ for the hydrolysis of 25 μm cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca²⁺. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.     

Interaction of cCMP with the cGK,cAK and MAPK Kinases in Murine Tissues

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-³²P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

Regulation of 6-phosphofructo-2-kinase from the citric-acid-accumulating fungus Aspergillus niger

Summary Phosphofructokinase 2 (PFK 2) was isolated from mycelia of the citric-acid-accumulating fungus Aspergillus niger, and partially purified by Trisacryl-Blue chromatography and Mono Q fast protein liquid chromatography. The appearance of a 96/94-kDa double band correlated with PFK 2 activity during purification. Purified PFK 2 had a half-life of 240 min at 4° C. The enzyme exhibited Michaelis-Menten type kinetics with respect to its substrates fructose-6-phosphate and ATP, required inorgaic phosphate for activity, and was only weakly inhibited by phospho(enol)pyruvate, AMP and citrate. The enzyme activity was not influenced by incubating partially purified PFK 2 preparations with ATP, MG²⁺ and the catalytic subunit of bovine heart protein kinase, although such treatment phosphorylated the 96/94-kDa protein. Consistently, treatment with alkaline phosphatase had no effect on PFK 2 activity. Also, no influence on PFK 2 activity was observed when cell-free extracts (containing A. niger protein kinases) from either glucose or citrate-grown mycelia were incubated with ATP and Mg²⁺ alone. It is concluded that, in A. niger, regulation of PFK 2 by phosphorylation/dephosphorylation does not occur, and this is related to the development of high glycolytic flow and citrate accumulation under conditions of supplying high sugar concentrations. Correspondence to: C. P. Kubicek     

A comparison of the uptake, metabolism, and action of cyclic adenine nucleotides in cultured hepatoma cells.

Intracellular radioactivity following incubation of HTC or RLC cells in [³H]cAMP exceeds that following incubation in either [³H]mono- or dibutyryl cAMP by 30-fold, yet little [³H]cAMP is found within the cells. Even at early times (30 min) the label derived from [³H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μm) result from incubation of both cell lines in either N⁶ mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP.Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N⁶ monobutyryl cAMP ? dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N⁶ monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C.The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [³H]cAMP) was of the order N⁶ monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio ? cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells.RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds.Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.     

Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues.

cAMP- and cGMP-dependent protein kinases are homologous proteins and are predicted to exhibit very similar three-dimensional structures. Their cyclic nucleotide binding domains share a high degree of amino acid sequence identity. cAMP- and cGMP-dependent protein kinases are activated relatively specifically by cAMP and cGMP, respectively; and a single alanine-threonine difference between cAMP- and cGMP-binding domains partially accounts for this specificity. Thus, it would be expected that cAMP and cGMP mediate separate physiological effects. However, owing in part to the lack of absolute specificity of either enzyme and to the relatively high level of cAMP or cGMP in certain tissues, it is also possible that either cyclic nucleotide could cross-activate the other kinase. Increases in either cAMP or cGMP cause pig coronary artery relaxation. However, only cGMP-dependent protein kinase specific cyclic nucleotide analogues are very effective in causing relaxation, and cAMP elevation in arteries treated with isoproterenol or forskolin activates cGMP-dependent protein kinase, in addition to cAMP-dependent protein kinase. Conversely, increases in either cAMP or cGMP cause Cl- secretion in T-84 colon carcinoma cells, and the cGMP level in T-84 cells can be elevated sufficiently by bacterial enterotoxin to activate cAMP-dependent protein kinase. These results imply specific regulation of cAMP- and cGMP-dependent protein kinases by the respective cyclic nucleotides, but either cyclic nucleotide is able to cross-activate the other kinase in certain tissues.     

Cyclic nucleotide content and protein phosphorylation during maturation of Spisula oocytes

Cyclic nucleotide levels in the oocytes of the surf clam Spisula solidissima were measured during germinal vesicle breakdown (GVBD) induced by fertilization. The level of cAMP and cGMP in untreated oocytes was 8.23 ± 0.95 and 4.89 ± 0.39 pmol/10⁶ oocytes. The ratio of cAMP to cGMP ranged from 1.5 to 2.0. The cAMP level in Spisula oocytes fluctuated after fertilization and before GVBD. The cGMP level showed minimal fluctuation, with a tendency to decrease initially followed by a subsequent rise to the basal level in a nonsynchronous manner. These changes were not statistically significant. There was a general increase in protein phosphorylation during the period after fertilization and before GVBD. The greatest increase occurred with proteins of estimated molecular weights of 52, 18, and 12 kD, analyzed by gel electrophoresis and autoradiography.     

Tissue Distribution,Developmental Changes and Some Catalytic Properties of Guanosine 3′,5′-Monophosphate-dependent Protein Kinase of Bombyx mori

The levels of guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase in the larval and pupal tissues of Bombyx mori were estimated. This activity was highest in the fat body of the female pupa. The enzyme showed a significant variation in activity during development of adult in female. Male silkworm gave less significant results. The cGMP-dependent kinase partially purified from the pupa could be activated by a high concentration of adenosine 3′,5′-monophosphate (cAMP) as reported for cGMP-dependent protein kinases from other sources. The nature of the enzyme thus activated and that of the enzyme activated by a low concentration of cGMP were found to be similar in several aspects. This indicates that the intrinsic activity of protein kinase from the silkworm pupa is independent of the kind of cyclic nucleotide as an activator.     

Cyclic nucleotide phosphodiesterases of rabbit renal cortex. Characterization of brush border membrane activities.

Cyclic nucleotide phosphodiesterase activity in brush border membranes, isolated from proximal tubule cells of the rabbit renal cortex, was investigated. Brush border cAMP phosphodiesterase activity was tightly bound to the membrane and was distinguished from the soluble phosphodiesterase activity of the renal cortex cytosol. Multiple forms of the brush border membrane cAMP phosphodiesterase activity, dependent on the concentration of substrate, were found. When assayed with 1 μm or 1 mm cAMP, activities differed in pH optimum, effects of various divalent cations, inhibition by metal ion chelators and reactivation by metals, thermolability, sensitivity to inhibitors and specificity.Renal brush border membranes also possessed cGMP phosphodiesterase activity. cAMP was a relatively poor inhibitor of the hydrolysis of 1 μm cGMP and the hydrolysis of 1 μm cAMP was virtually insensitive to cGMP. These findings suggest that the low substrate concentration-dependent cAMP phosphodiesterase was distinct from the low substrate concentration-dependent cGMP phosphodiesterase.Heat-stable effectors of phosphodiesterase activity were found in the renal cortex. One effector activated soluble cAMP phosphodiesterase. Activation was decreased by EGTA, enhanced by Ca²⁺ and diminished by preincubating the effector with proteolytic enzymes. The other heat-stable effector inhibited brush border membrane phosphodiesterase activity. Inhibition was unaffected by metal ions, unaffected by preincubating the effector with proteolytic enzymes, but diminished by preincubation with phospholipase C and neuraminidase.It is suggested that changes in the activity of the enzyme (or enzymes), which in turn controls, in part, the effective concentration of cAMP at its site (or sites) of action in the renal cell, may be significant in regulating hormonal-dependent transport in the proximal tubule.     

Roles of protein kinases in neurotransmitter responses in Xenopus oocytes injected with rat brain mRNA

Microinjection of rat brain mRNA in Xenopus oocytes induced acetylcholine, neurotensin, serotonin, and glutamate receptors in the cells. These receptors stimulate an intracellular reaction pathway, including G-protein activation, inositol trisphosphate (IP3) formation, and Ca2+-dependent Cl- channels. In the present study, we examined the roles of several protein kinases in these responses by means of inhibitors and activators of these kinases. Isoquinolinesulfonamides, inhibitors of protein kinases, caused no current responses and affected no receptor-mediated responses when injected into the oocytes at low doses (30-50 pmol), which inhibit cyclic nucleotide-dependent kinases or kinase C specifically, but abolished the receptor-mediated responses at a higher dose (300 pmol), which inhibit most protein kinases nonspecifically. Calmodulin inhibitors blocked the receptor-mediated responses strongly. Activation of cyclic nucleotide-dependent kinases or kinase C by injection of cAMP (or cGMP) or perfusion with phorbol esters caused no direct current responses but suppressed receptor-mediated responses. Current responses triggered by IP3 injection were not suppressed by these treatments. These results suggest that cAMP- (or cGMP-)dependent kinases or kinase C may not be involved in the pathway directly but may modulate it by inhibiting the initial part of the pathway (receptors, G-proteins, and/or phospholipase C), and they suggest that calmodulin may most likely be involved in the activation of Ca2+-dependent Cl- channels.     

Cyclic nucleotide-dependent protein kinases in airway smooth muscle

Because of the potential importance of cyclic nucleotide-dependent protein kinases in the regulation of airway smooth muscle tone, we have examined some of the characteristics of these enzymes in the soluble fraction of canine trachealis homogenates. In the absence of added cAMP, the heat-stable cAMP-dependent protein kinase inhibitor (PKI) abolished only a half of the 32P incorporation into mixed histones. The remaining activity appeared to be contributed by a cyclic nucleotide-independent enzyme. Phosphotransferase activity was enhanced 5-fold by 5 microM cAMP but only 70% of the cAMP-stimulated activity could be inhibited by PKI. The sensitivity of the cyclic nucleotide-dependent, PKI-resistant enzyme to cAMP, cGMP, and Mg2+ indicated that it was cGMP-dependent protein kinase. Because of the large amount of cyclic nucleotide-independent activity, and the ability of cAMP to activate cGMP-dependent protein kinase, the traditional "-cAMP/+cAMP" ratio did not provide an accurate assessment of the in vivo activation state of cAMP-dependent protein kinase. However, a modified assay was developed which allowed the precise measurement of cAMP-dependent, cGMP-dependent, and cyclic nucleotide-independent protein kinase activities. Using this new method, the cAMP-dependent protein kinase activity ratio of 0.239 in untreated trachealis strips was increased to 0.355 and 0.386 by prior exposure of the intact tissue to the smooth muscle relaxants isoproterenol and prostaglandin E2, respectively. The results of this study are consistent with the proposed role of cAMP-dependent protein kinase in the regulation of smooth muscle contractile function.