Early effects of TPA on protein kinase activity in murine thymocytes. Reduction of protein kinase C activity in the cytosol and increase of Ca2+ and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the particulate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca2+, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

Early effects of TPA on protein kinase activity in murine thymocytes : Reduction of protein kinase C activity in the cytosol and increase of Ca and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the pariculate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca²⁺, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

Redistribution of protein kinase C during mitogenesis of human B lymphocytes

G0 human tonsillar B-lymphocytes were stimulated to divide by the polyclonal mitogen Staphylococcus Aureus Cowan strain 1 (SAC) and by the combined use of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and the calcium ionophore ionomycin. The activities of protein kinase C, which requires Ca++ and phospholipid as co-factors, and a proteolytically cleaved form of this enzyme (protein kinase M), which is independent of calcium and phospholipid control, were determined in soluble and particulate fractions obtained from activated B cells. Treatment of G0 B cells with SAC or TPA together with ionomycin caused redistribution of protein kinase C from the soluble to the particulate fraction where the 80,000-Dalton protein kinase C was cleaved to give rise to a 50,000-Dalton form of the kinase which was also found in the cytoplasm. These data suggest that redistribution and proteolytic cleavage of protein kinase C are key signal transduction events in B cell mitogenesis.     

Modulation of Ca2+-activated, phospholipid-dependent protein kinase in platelets treated with a tumor-promoting phorbol ester

Incubation of human platelets with 12-0-tetradecanoylphorbol-13-acetate (TPA) caused a rapid decrease in soluble Ca2+, phospholipid-dependent protein kinase activity (protein kinase C) and an increase in protein kinase C associated with the particulate fraction. TPA also induced an increased activity of a Ca2+, phospholipid-independent protein kinase activity in both the soluble and the particulate fractions of platelets. This latter kinase eluted from DEAE cellulose columns at a higher salt concentration than protein kinase C, and was shown by Sephadex G-100 chromatography to have a MW of approx. 50,000 compared with an MW of 80,000 for protein kinase C. The data suggest that TPA treatment of platelets causes irreversible activation of protein kinase C by proteolysis of the enzyme to a form active in the absence of Ca2+ and phospholipid.     

Comparison of substrate recognition by protein kinase C (type III) between rat liver cytosolic and particulate fractions

1. Phosphorylation of rat liver endogenous substrates by protein kinase C (type III) was compared between cytosolic and particulate (mitochondria, microsomes and plasma membrane) fractions. 2. The rate and the maximum level of protein phosphorylation were several-fold higher in particulate fractions than in cytosolic fraction. 3. Protein phosphorylation in cytosolic fraction was dependent on both Ca2+ and phospholipid, but only Ca2+ was necessary in phosphorylation of particulate fractions. 4. These results suggest that protein kinase C (type III) has much more target proteins in particulate fractions rather than in cytosolic fraction and Ca2+ was important regulator in particulate protein phosphorylation.     

Increased expression of a high molecular weight (130 KD) protein kinase C isoform in a differentiation-defective ras-transfected keratinocyte line

The role of ras on protein kinase C (PKC) signaling was examined in two keratinocyte cell lines. Increasing the level of extracellular calcium from 0.15 mM to 1.0 mM induces some features of differentiation in the spontaneously immortalized HaCaT line, but fails to do so in a c-H-ras-transfected subline (ras-HaCaT). Raising extracellular calcium also induced a transient increase in membrane-associated PKC activity 5 min after calcium addition, in HaCaT, but not in the ras-HaCaT cells. Partial purification of PKC from the membrane/particulate fraction revealed the major isoform expressed in HaCaT to be an 80 KD species recognized by the anti-PKCα antibody. In ras-HaCaT, the major expressed isoform is a 130 KD species recognized by the PKCb? antibody. The kinase activity of the partially purified high molecular weight PKC is phospholipid dependent but calcium independent. Further evaluation of PKC in the HaCaT and ras-HaCaT membrane/particulate cell fraction by immunoblotting using affinity-purified antibodies against PKCα, b?, δ, ε and ζ revealed a 130 KD band reacting with the PKCδ antibody. Increased expression of this high molecular weight protein was observed in ras-HaCaT. Immunoprecipitation of PKC in ras-HaCaT using the PKCδ antibody also revealed a 130 KD species. Analysis of the PKCδ immunoprecipitate demonstrated a phospholipid, but not calcium-dependent kinase which autophosphorylated. These results suggest that the 130 KD protein may be a novel (calcium-independent) PKC (nPKC) isoform and increased expression in the rastransfected HaCaT may be a consequence of oncogenic ras expression. This 130 KD species may also play a role in the ras-associated inhibition of differentiation in HaCaT. © 1995 Wiley-Liss, Inc.     

Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in supernatant and particulate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine-rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30-60 min) of the cultures with 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not with 4 alpha-phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphorylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two-thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well-suited to respond to signals that activate phosphoinositide-linked receptors in brain.     

Angiotensin II but not potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerulosa cells

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was examined in bovine adrenal glomerulosa cells treated with angiotensin II or potassium. Protein kinase C was isolated from cytosol and from detergent-solubilized particulate fractions by DEAE-cellulose chromatography. A major peak of activity for both the soluble and particulate forms of adrenal glomerulosa protein kinase C was eluted at 0.05-0.09 M NaCl. The soluble and particulate forms were found to constitute about 95 and 5%, respectively, of the total enzyme activity in unstimulated cells. A second peak of kinase activity was eluted with 0.15-0.19 M NaCl, which was not dependent on the presence of phospholipids. Exposure of isolated cells for 20 min to 10(-8) M angiotensin II resulted in a decrease in cytosolic activity to 30-40% of control values, and in a corresponding increase in protein kinase C activity associated with the particulate fraction. This hormone-induced redistribution was found to be dose-dependent with an ED50 of 2 nM for angiotensin II, and it occurred rapidly, reaching a plateau within 5-10 min. It was prevented by the specific antagonist [Sar1,Ala8]angiotensin II. By contrast, stimulation with 12 mM KCl did not change the subcellular distribution of protein kinase C activity. These results suggest that redistribution of protein kinase C represents an early step in the post-receptor activation cascade following angiotensin II, but not potassium stimulation of adrenal glomerulosa cells.     

Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells

The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs.     

Membrane-associated protein kinases in phorbol ester-activated human polymorphonuclear leukocytes

Membrane-associated protein kinases in human polymorphonuclear leukocytes were studied. In unstimulated polymorphonuclear leukocytes the protein kinase C was predominantly present in the cytosol but in phorbol 12-myristate 13-acetate- (PMA-) activated cells a time and dose-dependent translocation of the kinase to the particulate fraction occurred. Two new protein kinase activities also appeared in the particulate fraction upon PMA activation. The one had a Mr of 40,000 and its activity was independent of phospholipids. The other (Mr 90,000) as partially activated by phospholipids, but separated from protein kinase C on DEAE-cellulose chromatography.     

Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.

In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.     

Examination of the role of the proteolytically-activated form of protein kinase C in the differentiation of human haemopoietic cells

Abstract. In neutrophils, the phorbol ester 12- O -tetrade-canoylphorbol-l3-acetate (TPA) induced the translocation of the Ca⁺⁺- and phospholipid-dependent protein kinase, protein kinase C (PK-C) from the soluble to the particulate fraction. At the same time there was a corresponding increase in the amount of Ca⁺⁺- and phospholipid-independent protein kinase activity recovered in the soluble fraction. This soluble Ca⁺⁺- and phospholipid-independent protein kinase presumably reflects proteolytic activation of the particulate associated PK-C. Bone marrow and undifferentiated HL-60 cells also translocated PK-C to the particulate fraction in response to TPA but did not accumulate the soluble Ca⁺⁺- and phospholipid-independent form of the enzyme. Similar results were obtained using HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO), recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) or la,25-dihydroxyvitamin D₃. There was also no significant change in either the number or time of expression of differentiation-specific cell surface antigens observed on HL-60 cells induced to differentiate with either DMSO, 1α,25-dihydroxyvitamin D3 or TPA in the presence of cyclosporin A, an agent reported to inhibit the proteolytic breakdown of PK-C to the Ca⁺⁺- and phospholipid-independent form. Likewise, cyclosporin A did not affect the rate or extent of differentiation of primary bone marrow cell cultures. These results suggest that the proteolytically activated and phospholipid-independent form of PK-C is probably not involved in haemopoietic cell differentiation.     

Evidence for increased synthesis as well as increased degradation of protein kinase C after treatment of human osteosarcoma cells with phorbol ester

Phorbol 12-myristate 13-acetate (PMA) induces time-dependent changes in protein kinase C subcellular distribution and enzymatic activity in the human osteosarcoma cell line SaOS-2. Short (less than 60 min) incubations with PMA caused decreased cytosolic enzyme activity and a concomitant increase in particulate protein kinase; after 3 h, particulate protein kinase C activity also declined to reach less than 10% of basal activity by 24 h (Krug, E., and Tashjian, Jr., A. H., (1987) Cancer Res. 47, 2243-2246). In order to determine whether the loss in enzyme activity was due to decreased enzyme protein, Western blot analyses were performed using a polyclonal antibody against protein kinase C raised in rabbits. This approach confirmed the previously reported time-related changes: 80-kDa immunoreactive protein kinase C initially translocated from the cytosol to the particulate cell fraction and later disappeared completely from the particulate fraction. Loss of protein kinase C enzymatic activity thus results from actual loss of the 80-kDa protein; we found no evidence for generation of a calcium/phospholipid-independent protein kinase C-like form of the enzyme. Membrane association was confirmed by immunoprecipitation experiments using [35S]methionine-labeled cells. Brief exposure to PMA caused a marked loss in the [35S]methionine-labeled cytosolic protein kinase C band and an increase in the labeled particulate band. Protein kinase C immunoprecipitated from cells treated with PMA for 14 h displayed an increase in [35S]methionine label despite a greater than 80% loss of enzyme activity. The high specific radioactivity of the remaining 80-kDa protein leads us to conclude that long term treatment with PMA causes an increase in the rate of protein kinase C synthesis accompanied by a still greater increase in the rate of enzyme degradation in SaOS-2 cells.     

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.     

Effect of membrane perturbation on protein kinase C activation: treatment with exogenous phospholipase C decreases translocation of enzyme to cellular membranes

Incubation of mouse epidermal HEL-37 cells with C. perfringens phospholipase C for 30 min caused a partial loss of protein kinase C activity after 30 min incubation. Essentially all the kinase activity was present in the cytosolic fraction of both control and treated cells, despite the continued hydrolysis of phospholipid by the exogenous phospholipase. At shorter incubation times with phospholipase C (5 and 10 min) an association of protein kinase with particulate material was observed, presumably reflecting the accumulation of diacylglycerol. It is proposed that further incubation with phospholipase C renders the plasma membrane unable to bind protein kinase C and that already bound enzyme is destroyed by proteolysis.     

The subcellular distribution of protein kinase Calpha, -epsilon, and -zeta isoforms during cardiac cell differentiation.

There is little information on the molecular events that control the subcellular distribution of protein kinase C during cardiac cell differentiation. We examined protein kinase C activity and the subcellular distribution of representatives of the "classical," "novel," and "atypical" protein kinase C's in P19 murine teratoma cells induced to undergo differentiation into cardiac myocytes by the addition of dimethylsulfoxide to the medium (Grepin et al., Development 124, 2387-2395, 1997). Differentiation was assessed by the presence of striated myosin, a morphological marker for cardiac cells. Addition of dimethyl sulfoxide to the medium resulted in the appearance of striated myosin by 10 days postincubation. Immunolocalization and Western blot studies revealed that a significant proportion of protein kinase Calpha, -epsilon, and -zeta were associated with the particulate fraction in P19 cells prior to differentiation. Differentiation into cardiac cells resulted in a translocation of protein kinase C activity from the particulate fraction to cytosol and localization of most of protein kinase Calpha, -epsilon, and -zeta to the cytoplasmic compartment. The total cellular protein kinase C activity was unaltered during differentiation. The translocation of protein kinase C activity during differentiation of P19 cells into cardiac myocytes was associated with a decrease in the levels of cellular 1, 2-diacyl-sn-glycerol. The cellular levels of phosphatidylserine and phosphatidylinositol did not change during differentiation. Addition of 1,2-dioctanoyl-sn-glycerol, a cell-permeant 1, 2-diacyl-sn-glycerol analog, reversed the differentiation-induced switch in the relative distribution of protein kinase C activity and dramatically increased the association of protein kinase Calpha with the particulate fraction. Addition of 1,2-dioctanoyl-sn-glycerol did not reverse the pattern of distribution for protein kinase Cepsilon or -zeta. The results indicate that protein kinase C activity and protein kinase Calpha, -epsilon and -zeta isoforms are redistributed from the particulate to the cytosolic fraction during differentiation of P19 cells into cardiomyocytes. The mechanism for the redistribution of protein kinase Calpha may be related to the reduction in the cellular 1,2-diacyl-sn-glycerol levels that accompany differentiation.     

A phorbol ester and phospholipid-activated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C

The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.     

Phytohemagglutinin treatment of T lymphocytes stimulates rapid increases in activity of both particulate and cytosolic protein kinase C

We have measured the activity of protein kinase C in particulate and cytosolic fractions prepared from lymphocytes following stimulation with phytohemagglutinin. Activity in the particulate fraction increased approximately three-fold within 5 min, and declined to nearly zero between 20 and 60 min. Cytosolic activity increased in a biphasic manner, with an initial increase at 5 min, a decline at 10 min, and a further increase by 20 min, which was sustained for at least 60 min. By contrast, 12-O-tetradecanoylphorbol-13-acetate caused a rapid translocation of protein kinase C from cytosol to the particulate fraction which was sustained for at least 1 h. The results suggest that agents, such as phytohemagglutinin, which both generate diacylglycerol and mobilize intracellular Ca2+ stores, result in changes in subcellular distribution and activity of protein kinase C which are different from those elicited by 12-O-tetradecanoylphorbol-13-acetate.     

Protein Kinase C Activity in Rat Brain Cortex

The procedure used to obtain cerebral tissue for analysis of protein kinase C (PKC) activity may affect the subcellular distribution of the enzyme. We compared different methods of tissue preparation and found that the proportion of PKC activity associated with the particulate fraction of the cerebral cortex was only 30% when the brain was frozen in situ while the animal was on life support or after decapitation followed by delayed freezing. Other methods of obtaining cerebral tissue resulted in 49-56% of the PKC activity in the particulate fraction. Freezing per se had no apparent effect on the activity or subcellular distribution of PKC. In addition, whenever the particulate PKC activity was high (greater than 48%), there was also a significant increase in the proportion of particulate protein (from 51 to approximately 63%, p less than 0.05).     

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 X 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 microgram/kg) resulted from an increase in independent protein kinase activity (-cyclic AMP) without a change in total protein kinase activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 microgram/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.