Soluble Protein Kinase Activity Research Articles

Memory impairment induced by chronic intracerebroventricular infusion of beta-amyloid (1–40) involves downregulation of protein kinase C

Signaling pathways underlying the cognitive deficit of the Alzheimer’s disease (AD) are not completely understood. Protein kinase C (PKC), a major neuronal protein plays a critical role in cellular signal transduction and it is known to be subjected to modulation in AD. We showed previously that, chronic infusion of β-amyloid (1–40) into rat cerebroventricle leads to deficit in spatial and non-spatial memory formation. As an attempt to identify the cellular correlates of the memory deficit, in the present study we investigated the PKC activation in different brain areas. Chronic infusion of β-amyloid (1–40) for 14 days into the rat cerebroventricle decreased the activity of soluble protein kinase C (PKC) in the hippocampus. Subcellular translocation of PKC to membrane fraction in hippocampal slices of rats treated with β-amyloid (1–40) was completely abolished under acute stimulation with 0.5 μM phorbol-dibutyrate (PDBu). We also reported a decreased affinity ( k D) for PDBu binding in the hippocampus, cerebral cortex and striatum. The total number of binding sites for PDBu ( B max) was increased, in the three brain areas analyzed on the day 14, but the changes were not statistically significant. Our data indicate that chronic accumulation of β-amyloid (1–40) into the rat brain reduced activation of PKC, effect that would substantially contribute to the memory deficit found in these animals.

Protein kinase C and cAMP‐dependent protein kinase regulate the neuronal differentiation of immortalized raphe neurons

These studies examined the extent to which protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) regulate the neuronal differentiation of the raphe-derived neuronal cell line, RN33B. A differentiation-specific 2.25-fold increase in soluble PKA activity was observed. Neither membrane-associated-PKA, -PKC, or soluble PKC activities changed concomitant with differentiation. The PKC activity was derived from PKC α, γ, ϵ, and θ isoenzymes. Activation of PKC inhibited the immunocytochemical expression of low and medium molecular weight neurofilament proteins, an effect due at least in part to decreased steady-state levels of protein. PKC activation also decreased glutamate immunoreactivity and increased cell number, protein synthesis, and bromodeoxyuridine uptake by 2.4-fold, 25%, and 32%, respectively. Coupled with the decrease in mature neuronal antigen expression, these data suggest that PKC activation inhibits neuronal differentiation by inducing proliferation. Inhibition of PKC markedly upregulated glutamate immunoreactivity. PKA activation potentiated the glutamatergic phenotype of RN33B cells, but inhibition of PKA was without effect on the expression of all neuronal antigens examined. Thus, both PKC and PKA regulate the differentiation of RN33B cells, although neither is absolutely necessary for expression of the differentiated neuronal phenotype. These results suggest the existence of parallel pathways regulating raphe neuronal differentiation. J. Neurosci. Res. 56:177–188, 1999. © 1999 Wiley-Liss, Inc.

Protein kinase C and cAMP-dependent protein kinase regulate the neuronal differentiation of immortalized raphe neurons.

These studies examined the extent to which protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) regulate the neuronal differentiation of the raphe-derived neuronal cell line, RN33B. A differentiation-specific 2.25-fold increase in soluble PKA activity was observed. Neither membrane-associated-PKA, -PKC, or soluble PKC activities changed concomitant with differentiation. The PKC activity was derived from PKC alpha, gamma, epsilon, and theta isoenzymes. Activation of PKC inhibited the immunocytochemical expression of low and medium molecular weight neurofilament proteins, an effect due at least in part to decreased steady-state levels of protein. PKC activation also decreased glutamate immunoreactivity and increased cell number, protein synthesis, and bromodeoxyuridine uptake by 2.4-fold, 25%, and 32%, respectively. Coupled with the decrease in mature neuronal antigen expression, these data suggest that PKC activation inhibits neuronal differentiation by inducing proliferation. Inhibition of PKC markedly upregulated glutamate immunoreactivity. PKA activation potentiated the glutamatergic phenotype of RN33B cells, but inhibition of PKA was without effect on the expression of all neuronal antigens examined. Thus, both PKC and PKA regulate the differentiation of RN33B cells, although neither is absolutely necessary for expression of the differentiated neuronal phenotype. These results suggest the existence of parallel pathways regulating raphe neuronal differentiation.

Activation of cAMP-dependent protein kinase in rat aorta by cAMP analogs is not correlated with relaxation.

The role of cAMP-dependent protein kinase (PKA) in the relaxation of vascular smooth muscle by cAMP analogs was studied. The analog N6,2'-O-dibutyryl-cAMP (dbu-cAMP) reduced KCl- and phenylephrine (PE)-induced tension in rat aortic rings in a dose-dependent fashion (10-100 microM). Conversely, incubation with 8-bromo-cAMP (8Br-cAMP; 10-100 microM) had very little effect on tension. The soluble and particulate PKA activity was determined in the analog-treated, PE-contracted tissue. Interference from extracellular analogs was eliminated by washout of analog prior to homogenization of the tissue for the PKA assay and by the addition of charcoal (10 mg/ml) to the homogenization buffer. The nonrelaxant analog 8Br-cAMP significantly increased the activity ratio of soluble PKA at a concentration of 30 microM and, in the particulate fraction, increased the activity ratio at 30- and 100 microM. Total PKA activity in the soluble fraction was significantly decreased by all concentrations of 8Br-cAMP used (10, 30, 100 microM). The relaxant analog dbu-cAMP had no significant effect at any concentration on catalytically active or total PKA activity levels. The only effect seen, at 30 microM, was an increase in the particulate activity ratio, and this was smaller than that seen with 8Br-cAMP. The basis for the alteration in PKA activity by 8Br-cAMP was a decrease in total soluble PKA activity as opposed to an increase in free catalytic PKA subunit. The fall in total activity was most likely due to adsorption by charcoal of catalytic subunit released from the 8Br-cAMP-activated holoenzyme. Thus, under the conditions used to assay PKA activity in cAMP-analog-treated vascular tissue, a decrease in total PKA activity represents an activation of the enzyme and, in fact, appears to be a better indicator of activation than changes in activity ratios. In either case, activation of soluble and particulate PKA by cAMP analogs did not correlate with relaxation of rat aortic rings.

The Interaction of Cations with Activity of Soluble Protein Kinase C from Mouse Brain

The interaction of some cations with the enzymatic activity of soluble protein kinase C was determined in order to elucidate whether protein kinase C can be activated by other metal cations besides Ca2+. Protein kinase C was activated by Ca2+ and Sr2+ having EC50 values of nearly 10 microM and 200 microM, respectively. Ba2+ likewise activated protein kinase C but was less potent. Co2+, Ni2+ and Mn2+ had no activating effects on the activity in the absence of Ca2+, but was slightly reduced in the presence of Ca2+ (0.5 mM). Cations with ionic radii close to Ca2+ (0.99 A) inhibited the activity irrespective of the absence or presence of Ca2+. The order of potency is as follows: Hg2+ greater than Cd2+ approximately Cu2+ much greater than Sm3+ greater than Tb3+ greater than La3+. Pb2+ and Zn2+, which showed a high affinity to SH-groups, as well as Hg2+, Cd2+ and Cu2+, which also inhibited the activity. Thus, among the ions investigated, the alkaline-earth ions Sr2+ and Ba2+ could be substituted for Ca2+, irrespective of ionic radii. The serious environmental pollutants such as Hg2+, Cd2+ or Pb2+ impaired the activity of protein kinase C probably due to SH-blocking.

Action of mercurials on activity of partially purified soluble protein kinase C from mice brain.

The enzymatic activity of soluble protein kinase C from mice brain was inhibited by mercuric chloride (II) (HgCl2) and organic mercurials, i.e. methyl mercury, phenyl mercury and p-chloromercuribenzoic acid (PCMB). The IC50 was 0.08 microM for HgCl2 and about 1 microM for organic mercurials. Sulfhydryl blocking reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) were less potent but nevertheless inhibited the enzymic activity of protein kinase C. The Hill coefficients of HgCl2, DTNB and NEM were close to unity whereas the values for organic mercurials were 1.3 to 1.5. The inhibition was of a non-competitive type with respect to H1 histone. 3H-PDBu binding activity was also inhibited by all of the reagents in a non-competitive manner. Mercurials apparently bind to sulfhydryl groups of protein kinase C to inhibit the enzymatic activity.

Characterization and androgenic regulation of soluble protein kinases and protein phosphorylation in rat ventral prostate gland

The soluble protein kinase activities for protamine and casein, the histone kinases modulated by cAMP or Ca 2+ and phospholipid, as well as the phosphorylation patterns of endogenous proteins were measured in rat ventral prostates from normal adults, castrates, and dihydrotestosterone-treated castrates. In normal prostate, the ratio of cAMP-dependent type I and II kinases was approximately 1:5. After a 3-week period of castration-induced regression, the concentrations of both enzymes were increased, but on a total organ basis, type I was decreased to 56%, while type II was reduced to 20% of normal levels. Casein kinase activity in unfractionated cytosol was not significantly altered by castration but when partially resolved into type I and II enzymes, there appeared to be a selective reduction in the type I component. In contrast, the total organ activities of protamine kinase or Ca 2+-activated, phospholipid-dependent kinase, two measures of protein kinase C enzyme, were significantly increased (64 and 71%, respectively) above sham controls in regressed organs of castrates. All of the castration-induced changes in protein kinases were restored toward normal by dihydrotestosterone treatment. Castration effects on protein kinase C and the cAMP-dependent kinases appeared to be manifest in the phosphorylation of endogenous proteins. Castration resulted in a qualitative shift in the cAMP-dependent phosphorylation patterns as measured by gel electrophoresis, with increases in four major bands and decreases in two others, whereas the Ca 2+-activated, phospholipid-dependent phosphorylation patterns were all enhanced. It is concluded that the androgenic regulation of protein kinase C differed qualitatively from that of other kinases, and its activation upon withdrawal of the androgenic stimulus may be involved in autophagic mechanisms in the prostate.

Platelet Ca 2+-activated, phospholipid-dependent protein kinase: Evidence for proteolytic activation of the enzyme in cells treated with phospholipase C

Incubation of human platelets with C . perfringens phospholipase C caused an increase in soluble protein kinase activity assayed in the presence of EGTA, and a decrease in Ca 2+/phospholipid-dependent protein kinase activity. Fractionation of extracts on DEAE-cellulose columns showed that phospholipase C treatment resulted in a new peak of protein kinase active in the presence of EGTA. On Sephadex G-100 chromatography this enzyme eluted as a single peak of protein kinase activity of MW about 50,000. An extract from untreated platelets eluted as a single peak of Ca 2+/phospholipid-dependent protein kinase of MW about 77,000. It was concluded that phospholipase C treatment resulted in the proteolysis of this latter enzyme to the lower MW form.

Regional distribution of calcium- and cyclic adenosine 3':5'- monophosphate-regulated protein phosphorylation systems in mammalian brain. II. Soluble systems

The regional distribution of phosphoproteins whose phosphorylation is regulated either by cyclic AMP or by calcium in combination with calmodulin or phospholipid has been investigated in particulate preparations from rat CNS. About 30 distinct phosphoproteins were observed. These phosphoproteins exhibited widely different patterns of regional distribution. Based upon distribution patterns, we have divided these phosphoproteins into three categories: category A, phosphoproteins found in all parts of the CNS in approximately equal amounts; category B, phosphoproteins which are widely distributed within the CNS but show large regional variations; and category C, phosphoproteins which show a highly restricted regional distribution. We have tentatively interpreted the results on particulate phosphoproteins in the following way: some are present in all or nearly all brain cells, others are present only in certain classes of brain cells, and still others have an even more limited distribution, being present in only a single type of brain cell. The regional distribution of particulate protein kinase activity was also examined. Calcium/calmodulin-dependent protein kinase activity had a marked regional distribution, whereas cyclic AMP-dependent protein kinase activity was more evenly distributed. Calcium/phospholipid-dependent protein kinase activity was barely detectable under the experimental conditions used. This investigation thus demonstrates striking differences in the regional distribution of particulate protein phosphorylation systems in mammalian brain. These regional differences may reflect highly specific functional roles for certain of these protein phosphorylation systems. Similar conclusions concerning cytosolic protein phosphorylation systems are described in the accompanying paper.

Hormonally specific phosphorylation of cardiac troponin I and activation of glycogen phosphorylase.

WE recently reported that PGE1 and isoprenaline have very different effects on the substrates of cyclic AMP-dependent protein kinase that regulate cardiac glycogen metabolism. Both agents elevated the cyclic" AMP content and the protein kinase activity ratio in isolated perfused rat hearts, but only isoprenaline caused changes in the activity of the substrates of protein kinase (presumably phosphorylation, causing activation of phosphorylase kinase and inactivation of glycogen synthase)1,2. Thus, the activation of soluble protein kinase appears insufficient to a cause alteration of the regulatory enzymes of glycogen metabolism. This suggests that the gly-cogenolytic responses to isoprenaline involve actions other than the activation of soluble protein kinase. We now report the effects of PGE1 and isoprenaline on another substrate of protein kinase, the inhibitory subunit of cardiac troponin (TNI). Since the phosphorylation of TNI may mediate the positive inotropic effects associated with β-adrenergic stimulation and cyclic AMP accumulation3, we felt that the hormonally specific expression of protein kinase activity might be used as a tool to determine whether similar specificity applies to the phosphorylation of TNI and to establish the functional relevance of that phosphorylation.

Properties of soluble cyclic AMP-dependent protein kinase activity of renal inner medulla

Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices. The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation.

Cyclic AMP-dependent and -independent protein kinases of the water mold, Blastocladiella emersonii

Protein kinase (ATP:protein phosphotrasferase, EC 2.7.1.37) and cyclic adenosine 3′,5′-monophosphate binding activities have been identified in zoospore extracts of the water mold Blastocladiella emersonii. More than 75% of these activities is found in the soluble fraction. Soluble protein kinase activity is resolved in three peaks (I, II and III) by DEAE-cellulose chromatography. Peak I is casein dependent and insensitive to cyclic AMP. Peak II is histone dependent and cyclic AMP independent; this enzyme is inhibited by the heat-stable inhibitor from bovine muscle, Peak III utilizes histone as substrate and is activated by cyclic AMP.

CYCLIC AMP‐DEPENDENT PROTEIN KINASE ACTIVITY AND SYNAPTOSOMAL PROTEIN PHOSPHORYLATION IN THE BRAINS OF AGED RATS

Abstract—The activity of soluble protein kinase and phosphorylation of endogenous synaptosomal proteins were studied in vitro, in the hippocampus and cerebral cortex of rats 3, 12, or 24 months of age. No between‐age differences in the activity of cyclic AMP‐dependent or independent protein kinase were detected in either brain region. The degree of stimulation by cyclic AMP and the apparent Ka, for cyclic AMP were similar at all stages. Cyclic AMP stimulated the phosphorylation of synaptosomal proteins from the cerebral cortex, hippocampus, caudate nucleus, and cerebellum of rats at all ages. There were no significant differences across age in the extent of phosphorylation of any membrane proteins in any brain region. The number and staining density of synaptosornal proteins separated by polyacrylamide gel electrophoresis were also similar at all ages. These studies indicate that the cyclic AMP‐dependent phosphorylation system in the rat brain does not change during advanced aging.

Cyclic nucleotide dependent protein kinase and the phosphorylation of endogenous proteins of retinal rod outer segments

Cyclic nucleotide dependent protein kinase has been extracted wiht Tris or Lubrol PX from purified rod outer segments (ROS) of bovine retina. The activity of the enzyme is unaffected by light but is stimulated by either cyclic guanosine 3',5'-monophosphate (cGMP) or cyclic adenosine 3',5'-monophosphate (cAMP). Most of the solubilized enzyme elutes from DEAE-cellulose with about 0.18 M NaCl (type II protein kinase). An endogenous 30,000 molecular weight protein of the soluble fraction of ROS as well as exogenous histone are phosphorylated by the protein kinase in a cyclic nucleotide dependent manner. The Tris-extracted enzyme can be reassociated in the presence of Mg2+ with ROS membranes that are depleted of protein kinase activity. The reassociated protein kinase is insensitive to exogenous cyclic nucleotides, and it catalyzes the phosphorylation of the membrane protein, bleached rhodopsin. While the soluble and membrane-associated protein kinases may be interchangeable, they appear to be modulated by different biological signals; soluble protein kinase activity is increased by cyclic nucleotides whereas membrane-bound activity is enhanced when rhodopsin is bleached by light.

Properties of epinephrine-induced activation of cardiac adenosine 3′,5′-monophosphate-dependent protein kinase

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (—cyclic AMP/+cyclic AMP) of 12 000 × g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 μg/kg) resulted from an increase in independent protein kinase activity (—cyclic 2 AMP) without a change in total protein observ activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 μg/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.

Pineal protein phosphorylation during serotonin N-acetyltransferase induction.

The activity of soluble protein kinase (ATP:protein phosphotransferase,EC 2.7.1.37) and pattern of nuclear protein phosphorylation was monitored in cultured rat pineal glands during the induction of serotonin N-acetyltransferase (acetyl-CoA:serotonin N-acetyltransferase;EC 2.3.1.5)by l-isoproterenol. A nuclear protein appears to be phosphorylated during the early stages of enzyme induction but is not phosphorylated at later stages of induction. This correlates well with the need for RNA synthesis associated with the induction process. The nuclear protein was also phosphorylated when the pineal glands were treated with dibutyryl 3':5'-cyclic AMP. The soluble protein kinase activity appeared to decline during mid-to-late stages of enzyme induction, but there was no concomitant increase in the particulate protein kinase activity.

ALTERATIONS OF CYCLIC NUCLEOTIDE‐RELATED ENZYMES AND ATPase DURING UNILATERAL ISCHEMIA AND RECIRCULATION IN GERBIL CEREBRAL CORTEX

Abstract— Several enzyme activities were determined in gerbil cerebral cortex during unilateral ischemia or in the post‐ischemic period following 1 h of ischemia. Adenylate cyclase and Na + ‐K + ‐activated ATPase showed essentially the same pattern. Neither enzyme changed during ischemia but the activities decreased on recirculation to 40–60% of right side control by 5 h. The ATPase had returned to control level by 20h; the adenylate cyclase by 7 days of recirculation. Particulate cyclic AMP‐dependent protein kinase in the ischemic left hemisphere decreased throughout the 6h of ischemia. It remained depressed in the first 5 h of the post‐ischemic period but returned to control by 20 h. The soluble protein kinase activity, the soluble cyclic AMP and cyclic GMP phosphodiesterase and the Mg2+ dependent ATPase did not change significantly during the ischemic or post‐ischemic periods. The results suggest that ischemia and/or recirculation may affect cellular membranes and membrane‐bound enzymes, in particular. Furthermore, the results imply that despite apparent metabolite recovery during the post‐ischemic period, enzymatic changes are occurring that may be important for both the quality of recovery and the response to further ischemic insult.

Inhibition of adenosine 3′:5′-monophosphate accumulation in white fat cells by short chain fatty acids, lactate, and β-hydroxybutyrate

The large increase in cyclic AMP accumulation by rat white fat cells seen in the presence of lipolytic agents plus methylxanthines and adenosine deaminase was markedly inhibited by lactate. However, lipolysis was unaffected by lactate. Octanoate, hexanoate, heptanoate, and p-hydroxybutyrate inhibited both cyclic AMP ac- cumulation and lipolysis by rat fat cells. The mechanism by which these acids inhibit lipolysis differs from that for long chain fatty acids such as oleate. Oleate directly in- hibited triglyceride lipase activity of homogenized rat adipose tissue. In contrast, octanoate, p-hydroxybutyrate, and lactate had no effect on triglyceride lipase activity. Hormone-stimulated adenylate cyclase activity of rat fat cell ghosts was inhibited by oleate and 4 mM octanoate but not by 1.6 mM octanoate, heptanoate, hexanoate, p-hydroxybutyrate or lactate. None of the acids affected the soluble protein kinase activity of rat adipose tissue. There was no stimulation by lactate, butyrate, p-hydroxy- butyrate, or octanoate of the soluble or particulate cyclic AMP phosphodiesterase activity of rat fat cell homogenates. The antilipolytic action of a short chain acid such as octanoate or hexanoate was not accompanied by any drop in total fat cell ATP. The mechanism by which lactate lowers cyclic AMP but not lipolysis remains to be established.

A plasmocytoma ribosome-associated protein kinase which phosphorylates a specific protein of the ribosomal KCl wash.

One ribosomal protein kinase activity and 3 soluble protein kinase activities have been identified in plasma cell tumors by DEAE-cellulose chromatography. We have shown phosphorylation in vivo and in vitro of a protein fraction from the ribosomal KCl wash which we have termed 'PPx fraction'. Phosphorylation of this protein fraction has been obtained in vitro with the ribosome-associated protein kinase. We have determined for the ribosomal protein kinase the following characteristics. 1. It is an Mg2+-dependent enzyme that transfers the gamma-phosphate from ATP into phosphoseryl and phosphothreonyl residues of the substrate. 2. It has a wide substrate specificity. Like the soluble protein kinases it catalyses the phosphorylation of several proteins like histone, phosvitin, casein and ribosomal proteins but it differs from the main soluble kinases (I, II) by the fact that it catalyses specifically the phosphorylation at least of one of the ribosomal KCl wash proteins. On dodecylsulfate-polyacrylamide gels this protein has a molecular weight of approximately 90000 and it is released from ribosomes under conditions commonly employed for extraction of initiation factors. 3. The ribosome-associated protein kinase is not stimulated by the addition of cyclic adenosine 3':5'-monophosphate. 4. KCl has no effect, NaCl has a weak effect on the phosphorylation, Mn2+ and Ca2+ are inhibitors. 5. ADP has been found to be a competitive inhibitor. 6. The maximum velocity of the ribosomal protein-kinase-catalysed reaction is 0.65 nmol of 32P incorporated in the KCl wash protein per min and per mg protein. 7. The apparent Km for the ribosomal KCl wash protein as substrate is 0.71 mg/ml and the Km for ATP is 94 muM. 8. The molecular weight of the ribosomal protein kinase, estimated by electrophoresis in polyacrylamide-dodecylsulfate gels, is 60000 and corresponds probably to a catalytic subunit.

Characterization of the protein kinases in a transplantable islet cell tumor of the syrian hamster

The protein kinase activities of a transplantable, insulin-producing hamster islet cell tumor were characterized using gel filtration, sucrose density gradient centrifugation and acrylamide gel electrophoresis. The post-microsomal supernatant fluid contains 70–80% of the protein kinase activity present in crude homogenates. A cAMP-dependent protein kinase, PK I ( M r 170,000), represents 25% of the soluble protein kinase activity assayed with protamine as substrate. It dissociates in the presence of cAMP into a cAMP-binding protein, R 2 ( M r 90,000) and a catalytic subunit C ( M r 33,000). The dissociation induced by cAMP seems to be facilitated by the addition of Mg 2+ and ATP. The regulatory subunit, R 2, changes its gel filtration pattern in the presence of 0.5 M NaCl suggesting dissociation into a smaller subunit, R 1 ( M r 44,000). By analogy with purified beef heart protein kinase (Erlichman et al., 1973) and skeletal muscle protein kinase (Beavo et al., 1974), an R 2C 2 structure is proposed for the cAMP-dependent insulinoma protein kinase, PK I. The presence in crude homogenates of a free cAMP-binding protein indistinguishable from the R 2 derived by dissociation of PK I, suggests that PK I is partially dissociated in vivo. A cAMP-independent (casein) kinase ( M r 210,000) elutes with PK I on columns of Sepharose 6B. Another cAMP-independent protein kinase, PK II ( M r 88,000), is the predominant form of soluble protein kinase accounting for approximately 75% of the soluble protein kinase activity detected using protamine as substrate. This cAMP-independent protein kinase changes its gel filtration pattern in the presence of 0.5 M NaCl giving rise to a form which appears to have the same M r (33,000) as the catalytic subunit of PK I. Studies comparing the catalytic subunit C of PK I with PK II and its salt-induced smaller molecular form demonstrate facile association of C with the cAMP-binding protein of purified bovine heart protein kinase to yield a hybrid holoenzyme, whereas PK II and its smaller form fail to recombine in this fashion. The 33,000 dalton forms derived from PK I (by cAMP) and PK II (by salt) also show different substrate specificities. It would appear, therefore, that pK II is a cAMP-independent protein kinase unrelated to PK I.