Phosphorylation of rabbit and human erythrocyte membranes by soluble adenosine 3':5'-monophosphate-dependent and -independent protein kinases.

Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase.     

Characterization of two tyrosine-specific protein kinases from pig spleen. Substrate-specific effect of autophosphorylation.

Spontaneously active tyrosine-specific protein kinases I and II (designated TyrK I and TyrK II) have been purified to electrophoretic homogeneity from a particulate fraction of porcine spleen based on an assay that used poly(4Tyr, Glu) as a substrate. SDS/polyacrylamide gels revealed a doublet of bands of about Mr 51,000 for TyrK I and two protein bands of Mr 55,000 and 54,000 for TyrK II. After incubation in the presence of [gamma-32P]ATP, the bands corresponding to both protein kinases contained phosphotyrosine. The two tyrosine protein kinases showed high activities with poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu) as substrates and lower activity with angiotensin II. Neither histone, phosvitin, casein nor bovine serum albumin were phosphorylated. Both protein tyrosine kinases were activated by millimolar concentrations of Mg2+ whereas Mn2+ was less effective. The effects of various polyanionic and polycationic substances depended on the nature of the peptide substrate. With poly(Tyr, 4Glu) as a substrate, the substances either inhibited the activities of TyrK I and TyrK II or had no effect. However, activation was observed with angiotensin II as substrate in the presence of polylysine, polyornithine, protamine sulfate, and heparin as effectors. When angiotensin II was used as substrate, activation also occurred by autophosphorylation, in parallel to the phosphate incorporation into the protein kinases. Activation by autophosphorylation was not observed with the synthetic peptide substrates, poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu).     

Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii

In previous work from this laboratory, a partially purified protein kinase from the soil amoeba Acanthamoeba castellanii was shown to phosphorylate the heavy chain of the two single-headed Acanthamoeba myosin isoenzymes, myosin IA and IB, resulting in a 10- to 20-fold increase in their actin-activated Mg2+-ATPase activities (Maruta, H., and Korn, E.D. (1977) J. Biol. Chem. 252, 8329-8332). A myosin I heavy chain kinase has now been purified to near homogeneity from Acanthamoeba by chromatography on DE-52 cellulose, phosphocellulose, and Procion red dye, followed by chromatography on histone-Sepharose. Myosin I heavy chain kinase contains a single polypeptide of 107,000 Da by electrophoretic analysis. Molecular sieve chromatography yields a Stokes radius of 4.1 nm, consistent with a molecular weight of 107,000 for a native protein with a frictional ratio of approximately 1.3:1. The kinase catalyzes the incorporation of 0.9 to 1.0 mol of phosphate into the heavy chain of both myosins IA and IB. Phosphoserine has been shown to be the phosphorylated amino acid in myosin IB. The kinase has highest specific activity toward myosin IA and IB, about 3-4 mumol of phosphate incorporated/min/mg (30 degrees C) at concentrations of myosin I that are well below saturating levels. The kinase also phosphorylates histone 2A, isolated smooth muscle light chains, and, to a very small extent, casein, but has no activity toward phosvitin or myosin II, a third Acanthamoeba myosin isoenzyme with a very different structure from myosin IA and IB. Myosin I heavy chain kinase requires Mg2+ but is not dependent on Ca2+, Ca2+/calmodulin, or cAMP for activity. The kinase undergoes an apparent autophosphorylation.     

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified.     

Open reading frame sso2387 from the archaeon Sulfolobus solfataricus encodes a polypeptide with protein-serine kinase activity

The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the "eukaryotic" protein kinase superfamily. sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures >or=60 degrees C, whereas phosphorylation of exogenous proteins was detectable at 37 degrees C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser(548), with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.     

Studies on protein kinases in two human rectocolic cell lines by polyacrylamide gel electrophoresis.Effect of the vasoactive intestinal peptide

Electrophoresis and subsequent assay of the enzyme directly onto the gel has allowed a rapid and quantitative characterization of the cyclic AMP-dependent and -independent histone kinases, protamine, phosvitin and casein kinases in HT 29 and HRT 18 cells. The technique has been applied to soluble extracts from cytoplasmic and nuclear fraction prepared in the presence and absence of neutral detergent. A more precise identification of these enzymes has been possible by analysing enzyme fractions obtained after ion-exchange chromatography of the above extracts. The protein kinase equipment of both cell lines was found to be identical (11 major components) but with different relative proportions of several enzymes. In cytoplasmic extracts: VIP activates only the type I, cytosolic, (band 4) and the type II, membrane-bound, (bands 6 and 8) cyclic AMP-dependent histone kinases. These enzymes account, respectively, for 34 and 55% of the total histone kinases in HT 29 and HRT 18 cells. The cyclic AMP-independent histone kinases (band 1,2,5 and 7) also phosphorylate protamine; band 5 was found 3o be much higher (4-fold) in HT 29 cells. In addition, two casein/phosvitin kinases have been identified in both cell lines with phosphorylating activity similar to the total histone kinases. In the nuclear extract two cyclic AMP-independent histone kinases have been found with electrophoretic mobility differing from the cytoplasmic enzymes. Also, two phosvitin/casein kinases specifically nuclear, due to their chromatographical and electrophoretical behaviour, have been characterized.     

Casein kinase activity in etiolated Cucumis sativus cotyledons

Two calcium- and light-dependent protein kinases have been reported in etiolated Cucumis sativus cotyledons (Vidal et al. 2007). In the present work, we studied casein kinase (CK) activity in etiolated cucumber cotyledons of in-gel and in vitro kinase assays, using specific CK inhibitors, and ATP and GTP as phosphate donors. Two proteins with CK activity were detected in both casein gels and autophosphorylation assays. One of them, with a molecular mass of approximately 36 kDa, showed biochemical CK1 characteristics: it was inhibited by specific CK1 inhibitors and only used ATP as phosphate donor. The second, with a molecular mass of approximately 38 kDa, had CK2 characteristics; it used both ATP and GTP as phosphate donors, was inhibited by all specific CK2 inhibitors, and was recognized by a polyclonal antibody directed against the α catalytic subunit of a CK2 from tobacco. The kinase activity of the CK2 detected in etiolated cucumber cotyledons showed circadian rhythmicity in both in vitro and in-gel casein phosphorylation and in autophosphorylation assays. Thus, our results suggest that the CK2 of approximately 38 kDa could be related to the circadian oscillator of C. sativus cotyledons.     

Purification of a yeast protein kinase sharing properties with type I and type II casein kinases

Cyclic nucleotide independent protein kinases preferring casein as in vitro substrates were resolved into four distinct species. Only one of the enzymes (CKII) was retained by DEAE-cellulose, whereas the three other enzymes (CKI-1, CKI-2, and CKI-3) were absorbed to CM-Sephadex, eluted with 250 and 600 mM NaCl, and fractionated by heparin-Sepharose chromatography. The casein kinase CKI-3 eluting at the highest NaCl concentration (550 mM) was purified to electrophoretic homogeneity by fast protein liquid chromatography. CKI-1 and CKI-2 correspond to mammalian type I casein kinase, because they bind to CM-Sephadex, they are monomeric enzymes of molecular weights below 50,000, they accept ATP exclusively (CKI-1) or predominantly (CKI-2) as phosphate donor, and they are either completely or relatively heparin insensitive. CKII corresponds to type II casein kinase due to its chromatographic properties, complex quaternary structure, nucleotide specificity (both ATP and GTP are phosphate donors), and heparin sensitivity. CKI-3 shares the following properties with type I casein kinases: it is retained by CM-Sephadex but not by DEAE-cellulose, and it consists of a monomeric protein having a molecular weight of 38,000. On the other hand, CKI-3 accepts both ATP and GTP with equal efficiency, and it is heparin sensitive (50% inhibition at 0.3 microgram/mL) like type II casein kinases. CKI-3 differs from the other three yeast casein kinases in requiring a low pH (5.5) and a high MgCl2 concentration (50 mM) for optimal activity. All four casein kinases phosphorylate their own catalytic protein at serine and threonine residues.     

Study of autophosphorylation of isoenzymes of cyclic AMP-dependent protein kinases.

Type I and type II cyclic AMP-dependent protein kinases, present in the cytosol from each of five rat and two bovine tissues, were separated from one another by DEAE-cellulose column chromatography in order to study their possible autophosphorylation. In each of the tissues studied, autophosphorylation of the regulatory subunit of the cyclic AMP-dependent protein kinase by the catalytic subunit could be demonstrated with the type II enzyme but not with the type I enzyme.     

Reversible phosphorylation of T-substrate by wheat germ, human erythrocyte, and rabbit skeletal muscle protein kinases

The reversibility of the reactions catalyzed by the wheat germ kinase and the cyclic AMP independent protein kinases isolated from human erythrocytes (casein kinases A and G) and rabbit skeletal muscle (casein kinases I and II) has been investigated. The reverse reaction requires ADP, Mg2+, phosphoprotein, and kinase and results in the formation of ATP from the phosphoprotein and ADP. The requirement for ADP in the wheat germ kinase and casein kinases II and G catalyzed reactions appears to be nonspecific. These kinases can also utilize GDP, IDP, and UDP as phosphoryl acceptors. Studies with the wheat germ protein T-substrate indicate that the phosphorylation of this protein substrate by the kinases is fully reversible. By contrast, the phosphorylation of phosvitin and casein is only partially reversible. Since the T-substrate is found to contain multiple phosphorylation sites and can serve as phosphoryl acceptor for the various kinases, the specificity of the phosphorylation of the substrate by the kinases is examined by way of the reverse reaction. The wheat germ kinase, casein kinase G, and casein kinase II appear to phosphorylate the same sites on the T-substrate as they are capable of completely dephosphorylating each other's 32P-T-substrate. Each of these kinases can catalyze the incorporation of 12 mol of 32P/48 000 g of T-substrate. In contrast, casein kinases A and I can incorporate only 6 mol of 32P/48 000 g of T-substrate. Studies on the reverse reactions suggest that these phosphorylation sites may be the same for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes

The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.     

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

The soluble protein kinase activities for protamine and casein, the histone kinases modulated by cAMP or Ca2+ and phospholipid, as well as the phosphorylation patterns of endogenous proteins were measured in rat ventral prostates from normal adults, castrates, and dihydrotestosterone-treated castrates. In normal prostate, the ratio of cAMP-dependent type I and II kinases was approximately 1:5. After a 3-week period of castration-induced regression, the concentrations of both enzymes were increased, but on a total organ basis, type I was decreased to 56%, while type II was reduced to 20% of normal levels. Casein kinase activity in unfractionated cytosol was not significantly altered by castration but when partially resolved into type I and II enzymes, there appeared to be a selective reduction in the type I component. In contrast, the total organ activities of protamine kinase or Ca2+-activated, phospholipid-dependent kinase, two measures of protein kinase C enzyme, were significantly increased (64 and 71%, respectively) above sham controls in regressed organs of castrates. All of the castration-induced changes in protein kinases were restored toward normal by dihydrotestosterone treatment. Castration effects on protein kinase C and the cAMP-dependent kinases appeared to be manifest in the phosphorylation of endogenous proteins. Castration resulted in a qualitative shift in the cAMP-dependent phosphorylation patterns as measured by gel electrophoresis, with increases in four major bands and decreases in two others, whereas the Ca2+-activated, phospholipid-dependent phosphorylation patterns were all enhanced. It is concluded that the androgenic regulation of protein kinase C differed qualitatively from that of other kinases, and its activation upon withdrawal of the androgenic stimulus may be involved in autophagic mechanisms in the prostate.     

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.     

Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity

The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C. V. C., (1987) Mol. Cell. Biol. 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme. The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated. The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila. The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt. The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q. A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis. The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme. The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin. Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme. When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified. From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates.     

Tissue distribution of casein kinases

The activities of casein kinases 1 and 2 in cytosol fractions prepared from 12 different rat tissues were compared. Casein kinase activities were detected in all tissues examined. Total casein kinase activities of lung, spleen, testis, and thymus were much higher than those of skeletal muscle, cardiac muscle, and adrenal gland. When activities of casein kinases 1 and 2 partially purified from lung, spleen, testis, and thymus prepared from 5 rats were compared, both total and specific activities of these kinases in testis were higher than those in the other tissues. These results indicate that testis is the most suitable tissue in rats for large-scale purification of casein kinase 1 as well as casein kinase 2.     

Site-specific phosphorylation of beta-casein by proetin kinases from rabbit reticulocytes

The B variant of beta-casein was phosphorylated with [gamma-32P]ATP using four different protein kinases isolated from rabbit reticulocytes. Casein was maximally phosphorylated by the individual protein kinase activities and subjected to chymotrptic digestion. The peptides were separated by a two-dimensional peptide fingerprinting technique, and the phosphorylated peptides were identified by autoradiography, The two phosphorylated peptides obtained from the action of casein kinase I were shown to have different migration patterns from those obtained with casein kinase II. The cAMP-regulated protein kinases had the same substrate specificity with beta-casein B, and the two phosphorylated peptides obtained using these enzymes were distinct from those phosphorylated by the cAMP-independent enzymes. Thus, the different protein kinases can be identified by substrate specificity using beta-casein.     

Tissue distribution of casein kinases

The activities of casein kinases 1 and 2 in cytosol fractions prepared from 12 different rat tissues were compared. Casein kinase activities were detected in all tissues examined. Total casein kinase activities of lung, spleen, testis, and thymus were much higher than those of skeletal muscle, cardiac muscle, and adrenal gland. When activities of casein kinases 1 and 2 partially purified from lung, spleen, testis, and thymus prepared from 5 rats were compared, both total and specific activities of these kinases in testis were higher than those in the other tissues. These results indicate that testis is the most suitable tissue in rats for large-scale purification of casein kinase 1 as well as casein kinase 2.     

Inhibition of casein kinase II by heparin

Casein kinase II, a cyclic nucleotide-independent protein kinase from rabbit reticulocytes, was shown to be inhibited by heparin. Heparin specifically inhibited the enzyme and had no effect on other protein kinases, including casein kinase I, the type I and II cAMP-dependent protein kinases, protease-activated kinase I, and the hemin-controlled repressor. Heparan sulfate was found to be 40-fold less effective than heparin towards casein kinase II; other acid mucopolysaccharides had little or no effect on the enzymatic activity. Steady state studies revealed that heparin acted as a competitive inhibitor with respect to the substrate, casein. A value of 20 ng/ml or about 1.4 nM was obtained for the apparent Ki. The inhibition was not reversed by ATP and varying the ATP and heparin concentrations in the assay only altered the maximum velocity.     

Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterization of their effects on enzyme activity

Three cyclic AMP-independent acetyl-CoA carboxylase kinases (A, B1 and B2) have been isolated from lactating rat mammary gland, using phosphocellulose chromatography, high performance gel filtration, and affinity chromatography on casein-Sepharose and phosvitin-Sepharose. These protein kinases have been identified with previously described kinases by the following criteria. Kinase A phosphorylates the same sites on rabbit mammary acetyl-CoA carboxylase as acetyl-CoA carboxylase kinase 2, which was originally described as a contaminant of rabbit mammary acetyl-CoA carboxylase purified by the poly(ethylene glycol)procedure. Kinase A will henceforth be referred to as acetyl-CoA carboxylase kinase-2. Kinase B1 has been identified with casein kinase II by its heparin sensitivity, elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. Kinase B2 has been identified with casein kinase I by its elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. The three kinases phosphorylate distinct sites on acetyl-CoA carboxylase. Phosphorylation by either casein kinase I or II does not affect enzyme activity. However, acetyl-CoA carboxylase kinase 2 inactivates acetyl-CoA carboxylase reversibly, in an identical manner to cyclic-AMP-dependent protein kinase, and phosphorylates sites located on identical peptides. Acetyl-CoA carboxylase kinase-2 can, however, be distinguished from the free catalytic subunit of cyclic-AMP-dependent protein kinase by its molecular mass, its substrate specificity, its elution behaviour on phosphocellulose, and its complete lack of sensitivity to the protein inhibitor of cyclic-AMP-dependent protein kinase. We also present evidence that phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase occurs directly and not via a bicyclic cascade system as proposed by other laboratories.