Cytosolic Protein Kinase Activity Research Articles

Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells.

In cultured vascular smooth muscle cells, angiotensin II (Ang II) stimulated a cytosolic protein kinase activity toward myelin basic protein (MBP) in a time- and dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate also increased the MBP kinase activity. Downregulation of protein kinase C by prolonged treatment of the cells with phorbol 12,13-dibutyrate markedly attenuated the Ang II- and PMA-induced MBP kinase activation. The Ang II- and PMA-stimulated MBP kinase activities were resolved almost equally into two distinct fractions on Mono-Q HR5/5 column chromatography (kinase 1 and kinase 2). The kinase assay in polyacrylamide gel revealed that apparent molecular masses of kinase 1 and kinase 2 were 40 and 45 kd, respectively. Microtubule-associated protein 2 also served as a substrate for both the kinases. Immunoblot analysis with an antiphosphotyrosine antibody suggested that both the kinases were tyrosine-phosphorylated during the action of Ang II. Phosphoamino acid analysis revealed that Ang II and PMA induced phosphorylation of both the kinases on serine/threonine as well as tyrosine residues. Phosphopeptide mapping patterns of kinase 1 and kinase 2 isolated from Ang II-stimulated cells were almost identical with those from PMA-stimulated cells. These results indicate that in vascular smooth muscle cells Ang II activates two species of MBP/microtubule-associated protein 2 kinases mainly through the protein kinase C-signaling pathway and suggest that tyrosine and serine/threonine phosphorylation may be involved in this process.

Glucagon, vasopressin and angiotensin all elicit a rapid, transient increase in hepatocyte protein kinase C activity.

Challenge of intact hepatocytes with one of the hormones vasopressin, angiotensin and glucagon or with the phorbol ester phorbol 12-myristate 13-acetate (PMA) led to a rapid increase in the activity of protein kinase C found in both cytosol and membrane fractions. Maximal activation by hormones occurred within 1-6 min of challenge of cells, after which activity declined. In membrane fractions protein kinase C activity return to basal levels some 15 min after exposure of cells to either angiotensin or glucagon. In cytosol fractions of cells challenged with hormones a second phase of activation ensued after about 10 min, with levels of protein kinase C activity remaining elevated above basal level 15 min afterwards. Activity changes elicited by PMA were rather different; it took about 15 min to achieve maximal activation of cytosolic protein kinase C activity. In membranes of cells challenged with PMA, an initial rapid and transient activation was followed by a sustained increase in activity occurring about 10 min after exposure of cells to this ligand. Only when hepatocytes were challenged with PMA was the translocation of protein kinase C from the cytosol to membrane fraction observed. The kinetics of PMA-induced translocation suggested that it accounted for the second phase of the increase in membrane protein kinase C activity which was unique to this ligand.

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K 0.5 of 10 μM) and maximum activation was observed at 50 μM arachidonate. Arachidonic acid did not affect the Ca 2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 μM) inhibited (> 90%) calmodulin-dependent protein kinase activity (K 0.5 = 50–100 μM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K 0.5 = 24 μM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50–100 μM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 μM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 μM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.

Enhanced membrane protein kinase C activity in myocardial ischemia.

The protein kinase activity in cytosol was similar in control, ischemic, and reperfused hearts; however, a 1.5-fold increase in membrane protein kinase activity was induced by ischemia and reperfusion. The H-7 inhibitable cytosolic protein kinase activity decreased by 40% with 30 min ischemia, while that of membrane fraction increased 1.8-fold. However, the CGS9343B inhibitable protein kinase activity in cytosolic fractions was unaffected by ischemia, while that of membrane increased by about 1.7-fold. These results suggest that myocardial ischemia is associated with enhanced protein kinase C and calmodulin-dependent kinase activities in membrane fraction. Furthermore, the results also suggest a translocation of protein kinase C activity from the cytosol to the membrane. Reperfusion of ischemic myocardium did not result in any further increase of protein kinase C and calmodulin-dependent kinase activities in the membrane. These enhanced protein kinase activities also resulted in an enhanced phosphorylation of endogenous membrane proteins. The creatine kinase released from the heart was increased by both ischemia and reperfusion. Therefore, these results suggest that biochemical cascades of reactions caused by enhanced membrane protein kinase C and calmodulin-dependent kinase activities may contribute to ischemic-reperfusion injury.

Arachidonic and cis-unsaturated fatty acids induce selective platelet substrate phosphorylation through activation of cytosolic protein kinase C

The ability of arachidonic acid and other fatty acids to induce phosphorylation of endogenous substrates and the role of protein kinase C in mediating these effects were examined. In a cell-free cytosolic system derived from human platelets, arachidonic, oleic, and other cis-unsaturated fatty acids induced a dose-dependent phosphorylation of several endogenous substrates. These substrates form a subset of phorbol ester-induced phosphorylations. Multiple lines of evidence suggested the direct involvement of protein kinase C in mediating fatty acid-induced phosphorylations. These observations suggest that arachidonic acid and other unsaturated fatty acids are capable of activating protein kinase C in a physiologic environment resulting in the phosphorylation of multiple endogenous substrates.

Diminished specific activity of cytosolic protein kinase C in sciatic nerve of streptozocin-induced diabetic rats and its correction by dietary myo-inositol.

The impaired Na(+)-K(+)-ATPase activity in peripheral nerve from diabetic rats is prevented by dietary myo-inositol (MI) supplementation in vivo and corrected by protein kinase C (PKC) agonists in vitro, suggesting that PKC may mediate the effects of nerve MI depletion on Na(+)-K(+)-ATPase activity. However, little is known about the effect of diabetes on PKC activity or peptide in rat peripheral nerve. Therefore, the effect of streptozocin-induced diabetes and dietary MI supplementation on the activity and distribution of PKC in rat sciatic nerve homogenates and cytosolic and particulate fractions was explored with histone phosphorylation assay and Western-blot analysis. PKC activity but not peptide was selectively decreased in the cytosolic fraction by streptozocin-induced diabetes, and this abnormality was partially corrected by dietary MI supplementation. These results suggest that altered MI metabolism may affect nerve PKC specific activity, and this alteration may play a role in reduced Na(+)-K(+)-ATPase activity and blunted regenerative response in diabetic nerve.

Diminished specific activity of cytosolic protein kinase C in sciatic nerve of streptozocin-induced diabetic rats and its correction by dietary myo-inositol

Diminished specific activity of cytosolic protein kinase C in sciatic nerve of streptozocin-induced diabetic rats and its correction by dietary myo-inositol

Alterations in Tyrosine Protein Kinase Activities Upon Activation of Human Neutrophils

Human neutrophils are phagocytic cells that can be activated by a variety of agonists to undergo a group of physiologic responses. This "stimulus-response" coupling is thought to be dependent on the phosphorylation of specific proteins. We have previously shown that, in addition to the widely distributed serine and threonine protein kinases, neutrophils contain tryosine protein kinase activity in the cell cytosol and particulate fractions. When neutrophils are treated with a variety of agents, phosphorylation of both cytosolic and particulate proteins on tyrosine residues occurs. Increases in tyrosine phosphorylation may be a result of increases in the activity of tyrosine kinases or a decrease in the activity of phosphotyrosine phosphatases. In this study, we have used a novel nondenaturing polyacrylamide gel electrophoretic method to demonstrate that treatment of human neutrophils with the chemotactic peptide FMLP or the phorbol ester phorbol myristate acetate induces a time- and concentration-dependent increase in the tyrosine protein kinase activity found in the cell cytosol and cell particulate fraction. The time course and concentration range over which these changes occur are similar to those seen for activation of the neutrophil oxidative burst and phosphorylation of proteins on tyrosine residues, suggesting that these events may be related.

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem Chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 n m) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 n m or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.

Tetanus toxin attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C in NG-108 cells

R. V. Considine, J. K. Bielicki, L. L. Simpson and J. R. Sherwin. Tetanus toxin attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C in NG-108 cells. Toxicon 28, 13–19, 1990.—Although the pathology of tetanus toxin poisoning has been linked to an inhibition of neurotransmitter release, the mechanism of this inhibition is unknown. The neuroblastoma × glioma hybrid cell NG-108 is an emerging model in which to study the biochemical effect of tetanus toxin on acetylcholine secretion. In differentiated as well as undifferentiated NG-108 cells, a 4 hr tetanus toxin (10 −8 M) pretreatment had no effect on basal levels of cyclic AMP or cyclic GMP. In addition, toxin pretreatment did not affect agonist induced increases in either cyclic nucleotide. Treatment of NG-108 cells for 4 hr with 10 −10 M tetanus toxin had no effect on the subsequently measured activity of cytosolic protein kinase C. However, a 4 hr pretreatment of undifferentiated or differentiated cells with tetanus toxin (10 −8 or 10 −10 M respectively) significantly attenuated the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C. Direct addition of tetanus toxin (10 −7−10 −10 M) to isolated protein kinase C did not alter the ability of the enzyme to phosphorylate histone protein. These results suggest that one manifestation of tetanus toxin poisoning may be a disruption in protein kinase C metabolism.

Hepatic calcium-binding protein regucalcin decreases Ca2+/calmodulin-dependent protein kinase activity in rat liver cytosol.

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on cytosolic Ca2+/calmodulin-dependent protein kinase activity was investigated. The increase in cytosolic Ca2+/calmodulin-dependent protein kinase activity with passage of incubation time was clearly prevented by the presence of regucalcin (1.0 microM). An appreciable effect of regucalcin was seen at 0.5 microM. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca2+ (0.25-1.0 mM). This increase was clearly blocked by the presence of regucalcin (1.0 microM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2.5-15 micrograms). In the presence of regucalcin (1.0 microM), trifluoperazine (50 microM), an antagonist of calmodulin, significantly decreased the cytosolic Ca2+/calmodulin-dependent protein kinase activity. These results suggest that regucalcin can regulate the Ca2(+)-calmodulin effect in liver cytosol.

Activation of hepatocyte protein kinase C by redox-cycling quinones.

The effects of quinone-generated active oxygen species on rat hepatocyte protein kinase C were investigated. The specific activity of cytosolic protein kinase C was increased 2-3-fold in hepatocytes incubated with the redox-cycling quinones, menadione, duroquinone or 2,3-dimethoxy-1,4-naphthoquinone, without alterations in particulate protein kinase C specific activity or Ca2+- and lipid-independent kinase activities. Redox-cycling quinones did not stimulate translocation of protein kinase C; however, activated protein kinase C was redistributed from cytosol to the particulate fraction when quinone-treated hepatocytes were exposed to 12-O-tetradecanoylphorbol 13-acetate (TPA). Quinone treatment did not alter cytosolic phorbol 12,13-dibutyrate (PDBu) binding capacity, and the cytosol of both control and quinone-treated hepatocytes exhibited a Kd for PDBu binding of 2 nM. Quinone-mediated activation of cytosolic protein kinase C was reversed by incubation with 10 mM-beta-mercaptoethanol, dithiothreitol or GSH, at 4 degrees C for 24 h. Furthermore, protein kinase C specific activity in control cytosol incubated in air increased by over 100% within 3 h; this increase was reversed by thiol-reducing agents. Similarly, incubation of partially-purified rat brain protein kinase C in air, or with low concentrations of GSSG in the presence of GSH, resulted in a 2-2.5-fold increase in Ca2+- and lipid-dependent kinase activity. In contrast with the effects of the redox-cycling quinones, when hepatocytes were treated with the thiol agents N-ethylmaleimide (NEM), p-benzoquinone (pBQ) or p-chloromercuribenzoic acid (pCMB), the cytosolic Ca2+- and lipid-dependent kinase activity was significantly inhibited, but the particulate-associated protein kinase C activity was unaffected. The Ca2+- and lipid-independent kinase activity of both the cytosolic and particulate fractions was significantly stimulated by NEM, but was unaffected by pBQ and pCMB. These results show that hepatocyte cytosolic protein kinase C is activated to a high-Vmax form by quinone-generated active oxygen species, and this effect is due to a reduction-sensitive modification of the thiol/disulphide status of protein kinase C.

Erythromycin and Roxithromycin Potentiate Human Neutrophil Locomotion In Vitro by Inhibition of Leukoattractant-Activated Superoxide Generation and Autooxidation

Erythromycin and especially roxithromycin (1.25-20 micrograms/mL) stimulated neutrophil migration in vitro. Both antibiotics selectively inhibited superoxide generation by neutrophils activated with the N-formylated leukotactic tripeptide FMLP, the calcium ionophore A23187 and the pharmacologic agent benoxaprofen, while the responses initiated by the tumor promotor PMA and opsonized zymosan were unaffected. Neutrophil autooxidation during exposure to FMLP was also decreased by both antibiotics. The antimicrobial agents did not scavenge superoxide. Likewise, the interactions of [3H]FMLP with specific receptors on neutrophils, FMLP-activated degranulation and intracellular calcium fluxes, the activity of cytosolic protein kinase C and the release of [3H]arachidonate from calcium ionophore-stimulated neutrophils were all unaffected by the antibiotics. Erythromycin and roxithromycin in particular appear to enhance neutrophil migration by an antioxidant mechanism that is not due to inhibition of transductional events involved in the activation of NADPH-oxidase or to oxidant scavenging properties.

Stimulation in vitro of Rabbit Erythrocyte Cytosol Phospholipid-dependent Protein Kinase Activity: A Novel Action of Thyroid Hormone

L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) at 10(-10) M stimulated phospholipid- and Ca2+-dependent protein kinase activity in rabbit red cell cytosol in vitro by 151 and 176%, respectively. Kinase of 30-fold greater specific activity, developed with 0.4 mM NaCl from cytosol applied to DEAE-cellulose, was also stimulated up to 2-fold by thyroid hormone. Hormone enhancement of kinase activity occurred after 60 min of incubation at 37 degrees C prior to enzyme assay. Thyroid hormone analogues triiodothyroacetic acid, 3,5-dimethyl-3'-isopropyl-L-thyronine, D-T3, D-T4, and 3,3',5'-L-triiodothyronine (reverse T3) were inactive. These results support a role for thyroid hormone endogenously in regulation of phospholipid-dependent protein kinase activity.

Activation of a cytosolic serine protein kinase by epidermal growth factor.

Exposure of A-431 cells to epidermal growth factor (EGF) results in a rapid enhancement (approximately 10-fold) of cytosolic serine protein kinase activity. The increase in serine kinase activity may be detected using a number of peptide and protein substrates. Enhancement of kinase activity occurs within 1 min of exposure of the cells to EGF and reaches a maximum in 5 min. Similar results were obtained with a variety of cell lines. We have partially purified the EGF-activated kinase from A-431 cells. It has an apparent molecular mass of approximately 100 kDa by gel filtration. One distinguishing property of the enzyme is its sensitivity to inhibition by micromolar quantities of polyarginine; polylysine has no effect. The EGF-activated kinase is unaffected by cyclic nucleotides, Ca2+/calmodulin, Ca2+/diolein/phosphatidylserine, or heparin. The enhancement of cytosolic serine kinase activity in A-431 cells appears to be an early event in cell "activation" by a number of biological response modifiers including EGF, bradykinin, 12-O-tetradecanoylphorbol-13-acetate, and histamine.

Effect of tolbutamide and glyburide on cAMP-dependent protein kinase activity in rat liver cytosol

Effect of tolbutamide and glyburide on cAMP-dependent protein kinase activity in rat liver cytosol

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

Insulin-stimulated protein kinase activity in rat skeletal muscle that phosphorylates ribosomal protein S6

Treatment of rats with a single high dose of insulin leads to rapid stimulation of cytosolic protein kinase activity in skeletal muscle that phosphorylates ribosomal protein S6. This stimulation is maximal within 15 minutes after insulin treatment, and the activity remains elevated for at least 90 minutes. The insulin-stimulated protein kinase activity elutes as two peaks from DEAE-Sepharose. Peak I elutes at 0.04-0.06 M KCl and is stimulated by insulin approximately 1.4-fold above the control. Peak II elutes at 0.09-0.11 M KCl and is stimulated 2.8-fold above the control. The peak II activity, which is most strongly stimulated by insulin, is resolved from cyclic AMP-dependent protein kinase on DEAE-Sepharose and appears to be distinct from protein kinase C. These results represent a novel finding of the stimulation of S6 kinase activity by insulin in skeletal muscle tissue in vivo.

Age-dependent changes in murine protein kinase and protease enzymes

Hormone responsiveness is mediated by signal transduction mechanisms involving second messengers, such as cAMP and Ca 2+, which regulate reversible changes in the phosphorylation state of proteins. During senescence individuals frequently exhibit a diminished responsiveness to hormones. We examined changes in enzymes involved in protein phosphorylation reactions that might account for this decreased adaptiveness in old mice, and observed the following post-maturational changes: (1) cAMP-dependent protein kinase (Pk-A) specific activity decreased in spleen cytosol and in the particulate fractions of lung, spleen and liver of 24-month-old mice as compared to 2-month-old mice. Splenic cytosolic Pk-A activity decreased by 18 months of age, while particulate activity decreased by 6 months; (2) The amount of 8-N 3-[ 32P]cAMP, a photoaffinity analog of cAMP, incorporated into Pk-A regulatory (R)-subunits from spleen and liver particulate fractions decreased, while photolabeling of R-subunit degradative products with this analog in heart and spleen cytosol increased. (3) Age-dependent increases in membrane-associated protease activities were found in all organs, along with a decrease in cytosolic lung calpain activity. these preteolytic changes may account for the enhanced R-subunit degradation and decreased Pk-A activities observed during senescence. (4) Age-dependent alterations in Ca 2+/phospholipid-dependent protein kinase (Pk-C) are organ specific: lung, liver, brain, and heart demonstrate no change in Pk-C activity, while spleen exhibits decreased activity. We hypothesize that these age-dependent alterations in kinase and proteolytic activities may be in part responsible for changes in cellular response to hormonal stimulation, differentiation signals, and antigen responsiveness during senescence.

Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes.

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.