Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells.

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.     

MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN AND MICROSOMAL FRACTIONS OF BOVINE BRAIN

Abstract— The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3',5'-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2 m -NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 m -NaCl for the microsomal enzyme. A peak with protamine and casein as substrate for the microsomal or myelin enzyme, respectively, was larger than that with histone as substrate for each enzyme. The first peak with histone as substrate on a DEAE–cellulose column appeared as two peaks on the Sepharose 6B column. The second peak with histone as substrate on DEAE–cellulose column was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and subunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6B column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size.
The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.     

Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.     

cAMP-dependent protein kinase isozymes with preference for histone H2B as substrate in mitochondria of bovine heart

Mitochondria from bovine hearts were fractionated by three different procedures and the fractions were characterized by marker enzymes. Highly purified outer membranes, membrane vesicles, and inner membranes, as well as two high-speed soluble fractions, were obtained. Azide (or oligomycin) resistant ATPase was not found to be a marker for outer membranes. The data were consistent with the association of the protein kinase activity with the soluble matrix of the mitochondria. Activity was highest with histone H2B as the substrate, with histone H1 next in preference. In contrast to the mitochondrial protein kinases studied previously, protamine, casein, and phosvitin were very poor substrates and there was no detectable phosphorylation of pyruvate dehydrogenase. Activity was stimulated by cAMP but not by cGMP, calmodulin, or phosphatidylserine--diolein, with or without Ca2+. Two cAMP-dependent isozymes were separated from the soluble fraction of the mitochondria by chromatography on DE-52 columns. Phosphorylation of histone H2B by the isozymes was inhibited by 98% by Kemptide.     

Protein kinase activity associated with Fc gamma 2a receptor of a murine macrophage like cell line, P388D1

The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc gamma 2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [gamma-32P]ATP. The autophosphorylation of Fc gamma 2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc gamma 2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (greater than 25 mM) or KCl (greater than 100 mM) or by a small amount of heparin (greater than 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc gamma 2a receptor used ATP as substrate with an apparent Km of 2 microM as well as GTP with an apparent Km of 10 microM. Prior heating (60 degrees C for 15 min) or treatment with protease (trypsin or Pronase) of Fc gamma 2a receptor complex almost totally abolished casein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.     

Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.     

Studies on plant gums. Proteases in neem (Azadirachta indica) gum

Proteolytic activity was detected in neem (Azadirachta indica) exudate gum when tested with casein and albumin as substrates. The enzyme activity was separated into two fractions by chromatography on TEAE-cellulose after EDTA treatment. Both the enzyme fractions were fairly stable to high temperatures and wide range of pH conditions. The pH optima were found to be around 6.5. Phenylmethyl sulphonylfluoride inhibited the activity of both the fractions. EDTA, Β-mercaptoethanol, tosylamide phenylethylchloromethylketone, tosyllysine chloroimethylketone,p-chloromercuribenzoate and dithiobis-2-nitrobenzoie acid did not affect the activity of the two enzyme fractions. The two fractions had no hydrolytic action on a variety of synthetic substrates tested.     

Nuclear protein kinase activities during the cell cycle of HeLa S3 cells

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.     

Modulation of protein phosphorylation by a factor purified from adipocytes.

1. A factor which modulates the activity of cyclic AMP-dependent protein kinase copurifies from rat adipocytes with an inhibitor of adenylate cyclase. Purification and stability studies suggest that both effects reside in a single factor previously referred to as a feedback regulator. 2. The magnitude and direction of the feedback regulator effect on cyclic AMP-dependent protein kinase activity was dependent on the concentration of feedback regulator and the concentration and type of protein substrate. Using histone type IIA as substrate, feedback regulator was inhibitory at low histone concentrations and stimulatory at high concentrations. Preincubation of protein kinase with feedback regulator resulted in inhibition at all histone concentrations. With some protein substrates, e.g. histone f2b and casein, inhibition was observed at all histone concentrations. 3. The stimulation of histone type IIA phosphorylation resulted from an increased V with no effect on either the apparent Ka for cyclic AMP or the Km for ATP. Time course studies suggest that feedback regulator increased the rate of phosphorylation without increasing the total number of phosphorylation sites. Increased histone phosphorylation was observed regardless of whether the cyclic AMP-dependent protein kinase was peak I or peak II (off Deae-cellulose), isolated from bovine or rabbit skeletal muscle or rat heart. A small stimulation was observed using cyclic GMP-dependent protein kinase. 4. These results indicate that feedback regulator can inhibit or stimulate protein kinase, an effect which is probably substrate directed, and depends on the reaction conditions. Whether feedback regulator modulated protein phosphorylation in vivo in addition to its inhibition of adenylate cyclase is unknown. However, stimulation of protein kinase activity in the presence of cyclic AMP is a valuable and rapid assay for monitoring feedback regulator fractions during purification procedures.     

Mechanism of activation of cyclic GMP-dependent protein kinase from rat liver by multiple modulators

A number of polyanionic compounds, including DNA, RNA and polyglutamate, were shown to exhibit protein kinase stimulatory modulator activity as they were required for cyclic GMP to stimulate the phosphorylation of various cationic substrates by rat liver cyclic GMP-dependent protien kinase. Anionic proteins (casein, phosvitin) were phosphorylated poorly by the enzyme and their phosphorylation was not stimulated by the stimulatory modulators. Studies of the mechanism of action suggest that the modulators interact directly with the substrates to form a complex which is a better substrate than free histone. The observed effect of modulator is complex as it depends on the ratio of modulator to histone and the resultant state of the complex formed (better or poorer substrate than free histone). The observed effect is also dependent on the properties of the histone substrate as Michaelis-Menten kinetics are not observed in the phosphorylation of arginine-rich histone in the absence or presence of cyclic GMP.     

Mechanism of activation of cyclic GMP-dependent protein kinase from rat liver by multiple modulators

A number of polyanionic compounds, including DNA, RNA and polyglutamate, were shown to exhibit protein kinase stimulatory modulator activity as they were required for cyclic GMP to stimulate the phosphorylation of various cationic substrates by rat liver cyclic GMP-dependent protein kinase. Anionic proteins (casein, phosvitin) were phosphorylated poorly by the enzyme and their phosphorylation was not stimulated by the stimulatory modulators. Studies of the mechanism of action suggest that the modulators interact directly with the substrates to form a complex which is a better substrate than free histone. The observed effect of modulator is complex as it depends on the ratio of modulator to histone and the resultant state of the complex formed (better or poorer substrate than free histone). The observed effect is also dependent on the properties of the histone substrate as Michaelis-Menten kinetics are not observed in the phosphorylation of arginine-rich histone in the absence or presence of cyclic GMP.     

Characterization of bovine brain calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.     

MODIFICATIONS IN SOLUBLE PROTEIN KINASE AND CYCLIC-AMP BINDING CAPACITY OF DEVELOPING RAT BRAIN

Histone and casein phosphoprotein-kinase activities were determined in rat brain soluble fraction at various stages of development. Cyclic AMP -independent or basal histone kinase activity increased, whereas cyclic AMP -dependent activity decreased in whole soluble fraction with the age. On the contrary, whole soluble cyclic AMP -dependent and -independent casein kinases activities did not show any difference during development. The percentage of activation by cyclic AMP of histone kinase activity and [³H] cyclic- AMP binding activity in the soluble fraction decreased markedly during development. By DEAE-cellulose chromatography the histone kinase was separated mainly into 4 peaks; the fourth peak was strongly stimulated by cyclic AMP . Stimulation by cyclic AMP was higher in the 4-day-old rat brains than in the 9- and 30-day-old. In the 9-day-old rats the ratio of cyclic AMP -dependent histone kinase in respect to the cyclic AMP -independent enzyme was higher than in 4- and 30-day-old rats. Casein kinase activities in the brains of 9- and 30-day-old rats were separated by DEAE-cellulose chromatography into three peaks of which the third one was stimulated by cyclic AMP . Little, if any, difference was observed for casein kinase during the development. These results suggest that brain histone and casein kinase are different enzymes:     

Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes.

Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.     

Relationship between RNA polymerase and protein kinase activities in rat mammary gland nuclei.

1. Extracts from rat mammary gland nuclei contain cyclic AMP -independent protein kinases which phosphorylate casein rather than histone. 2. A major increase in nuclear protein kinase activity occurred during late pregnancy and was maintained with the onset of lactation. 3. Two major peaks of activity were resolved by chomatography of nuclear extracts on DEAE-Sephadex; the first (NI) appeared in the void volume and the second (NII) was eluted by 0.05-0.12 M ammonium sulfate. Several other regions of lesser activity were also present. 4. Protein kinases in the cytosol 105,000 times g supernatant, precipitated by 70 percent ammonium sulfate, dialyzed against buffer, and chromatographed on DEAE-Sephadex, yielded a major components phosphorylated histone in preference to casein, and this was stimulated by cyclic AMP if histone was the substrate, but only the first (void volume) fraction was cyclic AMP-dependent when casein was used. 5. Most of RNA polymerases Ib and II, derived from the nucleolus and nucleoplasm, respectively, appeared in column fractions distinct from those containing the major NI and NII protein kinases. 6. Cyclic AMP altered the amount of RNA product synthesized by polymerases Ib and II, but the explanation for this is unknown. Due to their elution profiles and cyclic AMP-independence, protein kinases NI and NII are excluded from playing a catalytic role in these effects; participation of quantitatively minor protein kinases which co-elute with polymerase Ib and II is not yet excluded.     

Purification and characterization of a membrane-bound sialidase from pig liver

A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Characterization of Developmentally Regulated cAMP/Ca2+-Independent Protein Kinases from Dictyostelium discoideum

Cell-free extracts of the slime mold Dictyostelium discoideum were assayed for phosphorylating activity towards endogenous proteins and towards histone H1, casein and myelin basic protein (MBP). During development, protein kinase activity towards all of these substrates steadily increased and peaked between the aggregation and the pseudoplasmodial stages. Particulate-associated kinase activity was solubilized with 1% CHAPS, and separated into 300–400 kDa and ∼ 100 kDa components on Sephacryl S-300. The 300–400 kDa peak exhibited the most pronounced developmental increase in MBP phosphorylating activity. It was further fractionated on DEAE-Sephacel and heparin-Sepharose, and in each case, it coeluted with the histone H1 phosphorylating activity. The activity of this kinase was unaffected by cAMP and calmodulin, but it was reduced to 50% by ∼ 350 mM NaCl, 5 mM NaF and 40 μg polylysine/ml. The ∼ 100 kDa peak exhibited the most pronounced increase in casein kinase activity during development. Most of the casein phosphorylating activity did not bind to DEAE-Sephacel; it was distinct from casein kinase 2, which was not developmentally regulated. In parallel with these elevated kinase activities during development, there was increased in vitro phosphorylation of a number of Dictyostelium proteins, including two major phosphoproteins of 140 and 94 kDa.