Coelution of the type II holoenzyme form of cAMP-dependent protein kinase with regulatory subunits of the type I form of cAMP-dependent protein kinase

The types and subunit composition of cAMP-dependent protein kinases in soluble rat ovarian extracts were investigated. Results demonstrated that three peaks of cAMP-dependent kinase activity could be resolved using DEAE-cellulose chromatography. Based on the sedimentation of cAMP-dependent protein kinase and regulatory subunits using sucrose density gradient centrifugation, identification of 8-N3[32P]cAMP labeled RI and RII in DEAE-cellulose column and sucrose gradient fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Scatchard analysis of the cAMP-stimulated activation of the eluted peaks of kinase activity, the following conclusions were drawn regarding the composition of the three peaks of cAMP-dependent protein kinase activity: peak 1, eluting with less than or equal to 0.05 M potassium phosphate, consisted of the type I form of cAMP-dependent protein kinase; peak 2, eluting with 0.065-0.11 M potassium phosphate, consisted of free RI and a type II tetrameric holoenzyme; peak 3, eluting with 0.125 M potassium phosphate, consisted of an apparent RIIC trimer, followed by the elution with 0.15 M potassium phosphate of free RII. The regulatory subunits were confirmed as authentic RI and RII based upon their molecular weights and autophosphorylation characteristics. The more basic elution of the type II holoenzyme with free RI was not attributable to the ionic properties of the regulatory subunits, based upon the isoelectric points of photolabeled RI and RII and upon the elution location from DEAE-cellulose of RI and RII on dissociation from their respective holoenzymes by cAMP. This is the first report of a type II holoenzyme eluting in low salt fractions with free RI, and of the presence of an apparent RIIC trimer in a soluble tissue extract.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.     

Overexpression of the type II regulatory subunit of the cAMP-dependent protein kinase eliminates the type I holoenzyme in mouse cells

Mammalian tissues and cell lines express two major types of cAMP-dependent protein kinase, PKA-I and PKA-II, which can be distinguished at the molecular level by the presence of either type I or type II regulatory subunits in the holoenzyme. An expression vector for the mouse type II regulatory subunit (RII alpha) was transfected into ras-transformed NIH3T3 (R3T3) cells, which contain approximately equal amounts of both holoenzymes, PKA-I and PKA-II. In RII alpha-overexpressing R3T3 cells, PKA-II levels were increased, and the level of PKA-I declined. The decrease in PKA-I was dependent on the amount of RII alpha expressed, and at high levels of RII alpha expression, PKA-I was completely eliminated. In contrast, overexpression of the type I regulatory subunit (RI alpha) did not alter PKA isozyme levels. We propose that competition between RII alpha and RI alpha for a limited pool of catalytic subunit results in preferential assembly of PKA-II and that significant amounts of PKA-I are formed only if catalytic subunit is present in excess of the RII alpha subunit. The PKA-I isozyme, which is absent in untransformed 3T3 cells, is not essential for the transformed phenotype of R3T3 cells. RII alpha-overexpressing R3T3 cells that are devoid of PKA-I continued to exhibit a transformed phenotype including anchorage-independent growth. Overexpression of RII alpha provides a genetic approach that may prove useful in demonstrating specific functions for the two PKA isozymes in cAMP-dependent signal transduction pathways.     

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Reduced levels of cardiac cAMP-dependent protein kinase in spontaneously hypertensive rat

Cardiac cAMP-dependent protein kinases were compared between the spontaneously hypertensive rat and the age-matched normotensive Wistar-Kyoto rat by DEAE-cellulose chromatography, photoaffinity labeling with 8-N3[32P]cAMP, and Western blots using the antiregulatory and 125I-anticatalytic subunit antibodies. DEAE-cellulose chromatography revealed that the ratio of type I to type II cAMP-dependent protein kinase was 3:1 in the cytoplasmic soluble proteins from the heart of normotensive rat. In contrast, the ratio of type I to type II was 1:1 in the heart of hypertensive rat. Type I protein kinase was reduced by 3-fold in hypertensive rat compared to normotensive rat. The levels of type II protein kinase were similar in both normotensive and hypertensive rats. The ratio of regulatory subunits of type I (RI) to type II (RII) cAMP-dependent protein kinase was 2.5 in the soluble proteins from the heart of normotensive rat compared to a ratio of 0.62 for hypertensive rat. RI was reduced by 4-fold in hypertensive rat compared to normotensive rat. The decrease in RI from hypertensive rat was also demonstrated by photoaffinity labeling with 8-N3[32P] cAMP. Western blot analysis of the catalytic subunit revealed a 2-fold decrease in catalytic subunit (C) in the soluble proteins from the hypertensive rat compared to normotensive rat. These results show that the reduced level of activity of cardiac type I protein kinase in hypertensive rat was the result of a decrease in both the RI and C subunits, thus reducing the number of type I cAMP-dependent protein kinase holoenzyme molecules. Comparison of type I protein kinase from "prehypertensive" and "hypertensive" stages of hypertensive rat indicated that the type I protein kinase was reduced by 3-fold before an increase in the blood pressure was detectable. Cardiac type I protein kinase is predominantly associated with the cytoplasmic proteins in both the normotensive and hypertensive rats. The levels of RI, RII, and C associated with the membrane-solubilized proteins were not affected in the hypertensive rat. The levels of RII were similar in the brain tissue of normotensive and hypertensive rats, suggesting that the decrease in type I protein kinase is specific in hypertensive rat. In conclusion, a decrease in cardiac type I cAMP-dependent protein kinase may affect the degree of phosphorylation of cardiac regulatory proteins, thus impairing normal cardiac physiology in hypertensive rat.     

Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Delta(1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH(2) terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg(133) with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH(2)-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes.     

Quantitative changes in the catalytic and regulatory subunits of nuclear cAMP-dependent protein kinases type I and type II during isoproterenol-induced growth of the rat parotid gland

To quantify the cAMP-dependent protein kinases I and II in parotid gland nuclei independent of the enzyme activity, monospecific antisera against their subunits were applied in a sensitive enzyme immunoassay. About 3% of total catalytic subunit in the homogenate was found in the isolated nuclei. During beta-agonist-induced proliferation of the parotid gland the nuclear concentration of catalytic and regulatory subunits changed. Related to the number of nuclei, the catalytic subunit and the regulatory subunit RI increased about 3-fold whereas the regulatory subunit RII remained unchanged.     

Neuronal microtubule-associated protein 2D is a dual a-kinase anchoring protein expressed in rat ovarian granulosa cells

A-kinase anchoring proteins (AKAPs) function to target protein kinase A (PKA) to specific locations within the cell. AKAPs are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an in vitro overlay assay. We previously showed that follicle-stimulating hormone (FSH) induces the expression of an 80-kDa AKAP (AKAP 80) in ovarian granulosa cells as they mature from a preantral to a preovulatory phenotype. In this report, we identify AKAP 80 as microtubule-associated protein 2D (MAP2D), a low molecular weight splice variant of the neuronal MAP2 protein. MAP2D is induced in granulosa cells by dexamethasone and by FSH in a time-dependent manner that mimics that of AKAP 80, and immunoprecipitation of MAP2D depletes extracts of AKAP 80. MAP2D is the only MAP2 protein present in ovaries and is localized to granulosa cells of preovulatory follicles and to luteal cells. MAP2D is concentrated at the Golgi apparatus along with RI and RII and, based on coimmunoprecipitation results, appears to bind both RI and RII in granulosa cells. Reduced expression of MAP2D resulting from treatment of granulosa cells with antisense oligonucleotides to MAP2 inhibited the phosphorylation of cAMP-response element-binding protein. These results suggest that this classic neuronal RII AKAP is a dual RI/RII AKAP that performs unique functions in ovarian granulosa cells that contribute to the preovulatory phenotype.     

cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.     

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.     

Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.     

In situ reassociation of the regulatory and catalytic subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase isoenzymes in AtT20 cells

Dissociation and reassociation of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases I and II were studied in intact AtT20 cells. Cells were stimulated with 50 microM forskolin to raise intracellular cAMP levels and induce complete dissociation of R and C subunits. After the removal of forskolin from the incubation medium cAMP levels rapidly declined to basal levels. Reassociation of R and C subunits was monitored by immunoprecipitation of cAMP-dependent protein kinase activity using anti-R immunoglobulins. The time course for reassociation of R and C subunits paralleled the loss of cellular cAMP. Total cAMP-dependent protein kinase activity and the ratio of protein kinase I to protein kinase II seen 30 min after the removal of forskolin was the same as in control cells. Similar results were seen using crude AtT20 cell extracts treated with exogenous cAMP and Mg2+. Our data showed that after removal of a stimulus from AtT20 cells inactivation of both cAMP-dependent protein kinase isoenzymes occurred by the rapid reassociation of R and C subunits to form holoenzyme. Our studies also showed that half of the type I regulatory subunit (RI) present in control cells contained bound cAMP. This represented approximately 30% of the cellular cAMP in nonstimulated cells. The cAMP bound to RI was resistant to hydrolysis by cyclic nucleotide phosphodiesterase but was dissociated from RI in the presence of excess purified bovine heart C. The RI subunits devoid of C may function to sequester cAMP and, thereby, prevent the activation of cAMP-dependent protein kinase activity in nonstimulated AtT20 cells.     

Modulation of soluble ovarian adenosine 3',5'-monophosphate-dependent protein kinase activity during prepubertal development in the rat. II. Evaluation of the catalytic subunit inhibitor activity

In a previous report on the ontogeny of the ovarian adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity during prepubertal development of the rat, we concluded that the 4-fold decline in cAMP-dependent protein kinase activity observed in ovaries of 21- to 23-day-old rats was due to the presence of a heat-labile inhibitor in the ovarian extracts (Hunzicker-Dunn et al., 1984). We developed an assay for this ovarian kinase inhibitor activity that was based on the observation that ovarian cytosol added to an exogenous catalytic subunit of cAMP-dependent protein kinase caused a time-dependent and ovarian cytosol protein concentration-dependent inhibition of exogenous catalytic subunit phosphotransferase activity. The present studies were conducted to evaluate the basis for this catalytic subunit inhibitor present in soluble rat ovarian extracts of prepubertal-aged rats. This inhibitor activity was absent from cytosol extracts of rat corpora lutea, rat liver, rabbit follicles, and rabbit corpora lutea. Inhibitor activity present in rat ovarian cytosol was not attributable to insufficient levels of the phosphorylation substrate Kemptide. Inhibitor activity was also not related to the presence of the large amount of catalytic subunit-free regulatory subunit of the cAMP-dependent protein kinase present in ovarian extracts of late juvenile-aged rats. Inhibitor activity, however, did correlate with an endogenous adenosine triphosphatase (ATPase) activity that reduced assay ATP concentrations below levels needed to accurately measure phosphotransferase activity, despite the presence of sodium fluoride (an ATPase inhibitor) and ATP concentrations 5- to 15-fold greater than the Km of the kinase for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.     

The phosphoform of the regulatory subunit RII of cyclic AMP-dependent protein kinase possesses intrinsic topoisomerase activity

The phosphoform of the type II regulatory subunit (phospho-RII-cAMP) of cAMP-dependent protein kinase from rat liver was found to possess intrinsic topoisomerase activity towards several DNA substrates such as phi X174, pBR322, SV40, and M13. Like the type I topoisomerases from several eukaryotic cells, phospho-RII X cAMP can relax both positive and negative superhelical turns of phi X174 DNA. Topological isomers with a decreasing number of superhelical turns can be identified as transient products. Conditions under which phospho-RII X cAMP relaxes superhelical phi X174 DNA lead to transient formation of a DNA-phospho-RII X cAMP complex via DNA strand breakage and covalent attachment of the DNA to a tyrosine residue of phospho-RII X cAMP via a phospho-RII X cAMP depends on the presence of cAMP and is altered by changes in the degree of phosphorylation of RII. Both dephosphorylation and removal of cAMP from phospho-RII X cAMP abolish its topoisomerase activity.     

Characterization of the protein kinase activities of human platelet supernatant and particulate fractions.

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.