Purification and characterization of a 400-kDa nonhistone chromatin protein that serves as an effective phosphate acceptor for casein kinase II from Ehrlich ascites tumor cells

A nonhistone chromatin protein (NHCP) has been purified to homogeneity from a 0.5 M NaCl extract of Ehrlich ascites tumor cell (EAT cell) nuclei as a phosphate acceptor for casein kinase II using ion-exchange column chromatographies and Sephacryl S300 gel filtration. The purified NHCP (approximate Mr = 400,000) was found to be a tetramer of an Mr = 98,000 polypeptide (pI = 6.9) and to have high contents of glycine (15%) and serine (11.6%). This protein (designated as 400-kDa NHCP) was highly phosphorylated by casein kinase II (Mr = 130,000), but not by histone kinase. Casein kinase II phosphorylated only seryl residues of the purified 400-kDa NHCP. The NHCP bound with DNA, but not with RNAs, and the DNA binding ability of the protein was reduced when it was phosphorylated by casein kinase II. Moreover, we found that (a) the 400-kDa NHCP is present in large quantities in malignant mouse cells, such as EAT, EL-4, and Meth-A cells, but only slightly in normal tissues and cells; (b) the protein level is rapidly increased when mouse lymphocytes are treated with recombinant interleukin 2 (T cell growth factor) or concanavalin A; and (c) the kinase responsible for the 400-kDa NHCP phosphorylation in the chromatin of various mouse cells is a casein kinase II. These experimental results suggest that the 400-kDa NHCP acts as an effective phosphate acceptor for casein kinase II at the chromatin level and that an increased phosphorylation of the protein by the kinase may be implicated in the progress of cell differentiation and proliferation.     

Quantitative determination of nucleic acids in brown and white adipose tissue

The low-molecular-weight soybean antiprotease C-II is specifically phosphorylated by a cAMP-independent protein kinase (Casein kinase TS) much more readily than casein itself. Unlike the routinely used substrates casein and phosvitin, C-II is not affected either by casein kinases S or by the cAMP-dependent protein kinase. It can be successfully employed therefore for the direct and reliable evaluation of casein kinase TS within preparations still contaminated by other protein kinases. Crude preparations of soybean antiproteases can replace C-II as specifically phosphorylatable substrate of casein kinase TS, though displaying a much lower phosphorylation efficiency.     

Isolation and characterization of casein kinases I and II from rabbit liver. Analysis of their effects on protein biosynthesis in the cell

Two cAMP-independent protein kinases isolated from rabbit liver extracts phosphorylate casein far more effectively than histones. The first protein kinase consists of one polypeptide chain (Mr = 37,000), utilizes exclusively ATP and is not inhibited in the presence of low heparin and RNA concentrations. The second protein kinase consists of three subunits (Mr = 42,000, 40,000 and 25,000 Da), utilizes both ATP and GTP and is inhibited by low heparin and RNA concentrations. The latter enzyme has Mr approximately 140,000 Da and possesses a polyanion-binding activity. These characteristics allow to relate the above enzymes to casein kinases I and II, respectively. Injection of casein kinase I into frog oocytes results in the inhibition of the rate of amino acid incorporations into the soluble and detergent extractable proteins. Casein kinase II has no effect on the amino acid incorporation into the recipient oocytes.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Biosynthesis and posttranslational modifications of protein kinase C in human breast cancer cells

Several forms of protein kinase C with molecular masses of 74-, 77-, and 80-kDa were detected in subcellular fractions of human breast cancer MDA-MB-231 cells which express the alpha-type protein kinase C. Several lines of evidence indicated that the 74-kDa is the precursor of the 77- and 80-kDa protein kinase C forms. (i) Pulse-labeling experiments revealed that protein kinase C is synthesized on membranes as a 74-kDa protein that can be chased into the 77- and the 80-kDa protein kinase C forms. (ii) The primary translation product of protein kinase C displayed an apparent molecular size of 74-kDa as determined by in vitro translation of poly(A)+ RNA from MDA-MB-231 cells. (iii) Incubation with serine/threonine-specific protein phosphatases (potato acid phosphatase and phosphatase 1 or 2A) resulted in the complete dephosphorylation of the 77-kDa to the 74-kDa protein kinase C form. Protein kinase C appears to be synthesized in membranes as an unphosphorylated and presumably inactive 74-kDa form that is converted into the active 77- and 80-kDa protein kinase C by post-translational modification involving at least two phosphorylation steps. The first phosphorylation is probably achieved by a specific, yet unidentified, "protein kinase C kinase" since the 74-kDa protein kinase C species did not undergo autophosphorylation and was neither a substrate for the purified protein kinase C, S6 kinase, phosphorylase kinase, casein kinase II, nor for the catalytic subunit of cAMP-dependent protein kinase. Except for phosphorylase kinase and the catalytic subunit of the cAMP-dependent protein kinase, phosphorylation of the 77-kDa protein kinase C form with purified protein kinase C (autophosphorylation), S6 kinase or casein kinase II shifted the molecular mass of the 77-kDa protein kinase C to 80-kDa. Prolonged exposure of MDA-MB-231 cells to phorbol 12-myristate 13-acetate not only leads to a complete down-regulation of protein kinase C activity but also to an accumulation of 74-kDa protein kinase C due to a retarded conversion of the 74-kDa into the 77- and 80-kDa protein kinase C forms in these cells. Our data indicate that tumor promoters additionally interfere with the posttranslational processing that converts the 74-kDa protein kinase C precursor into the 77- and 80-kDa forms of the enzyme.     

Phosphorylation in vitro of fibrinogen from three mammalian species with four different protein kinases

Human, dog, and rabbit fibrinogen served as substrates for calcium-activated, phospholipid-dependent protein kinase, cAMP-dependent protein kinase, casein kinase TS, and casein kinase S. The chains of phosphorylated fibrinogen were separated by polyacrylamide gel electrophoresis and the phosphorylation patterns, obtained on autoradiography of the gels, were found to be characteristic for each of the four protein kinases. Dog, and even more so rabbit, fibrinogen was phosphorylated more rapidly than human fibrinogen by calcium-activated, phospholipid-dependent protein kinase and by casein kinase TS. Dog fibrinogen was not a good substrate for cAMP-dependent protein kinase. The rate of phosphorylation with casein kinase S did not differ very much between the fibrinogens of the three species. In most cases the A alpha-chain was most rapidly phosphorylated. However, in dog fibrinogen incubated with casein kinase TS the B beta-chain was most rapidly phosphorylated. A substantial part of this phosphate seemed to be incorporated as phosphorylthreonine into fibrinopeptide B. In human fibrinogen incubated with the casein kinase TS preparation the gamma-chain as well as the A alpha-chain appeared to be phosphorylated.     

Characterization in Mammalian Brain of a DARPP-32 Serine Kinase Identical to Casein Kinase II

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein of Mr 32,000, is phosphorylated in vitro by casein kinase II at a site which is also phosphorylated in intact cells. In the present study, we show that a protein kinase activity, present in caudate-putamen cytosol, phosphorylates DARPP-32 on a seryl residue located on the same thermolytic peptide that is phosphorylated by purified casein kinase II. This DARPP-32 serine kinase was indistinguishable from casein kinase II on the basis of a number of biochemical criteria. Excitotoxic lesions of the caudate-putamen and immunocytochemistry revealed the presence of casein kinase II in the medium-sized striatonigral neurons which are known to contain DARPP-32. Casein kinase II activity was high in all rat brain regions studied, and casein kinase II-like immunoreactivity was detected in most brain neurons, although some neuronal populations (e.g., cortical pyramidal cells and large striatal neurons) were stained more intensely than others. In rat caudate-putamen, 45% of the total casein kinase II activity was in the cytosol and 20% in the synaptosomal fraction. In mouse cerebral cortex and caudate-putamen, casein kinase II activity was high at embryonic day 16, and remained elevated during development. In addition to DARPP-32, several major substrates for casein kinase II were observed specifically in brain, but not in liver extracts. The high activity of casein kinase II in brain from the embryonic period to adult age and the existence of a number of specific substrates suggest that this enzyme may play an important role in both developing and mature brain, possibly in modulating the responsiveness of target proteins to various extracellular signals.     

cAMP-independent casein kinase mediates phosphorylation of many T3-dependent phosphoproteins in rat liver cytosol

Administration of T3 (20 micrograms/100 g BW) for 3 days increases phosphorylation of several proteins in rat liver cytosol in vitro. To help elucidate the mechanism of T3-induced phosphorylation, we studied which protein kinase(s) mediate phosphorylation of endogenous cytosolic proteins. Five different protein kinases were obtained by DEAE+ cellulose column chromatographic fractionation of liver cytosol. When their ability to phosphorylate heat-inactivated cytosol was investigated, casein kinase, a cAMP independent protein kinase, showed the strongest effect. Casein kinase, purified by phosphocellulose chromatography, phosphorylated more than 10 cytosolic proteins. Several T3-dependent (and cAMP independent) phosphoproteins were included among these. One protein with Mr 39 X 10(3), of which phosphorylation is stimulated by T3 within five hours after injection, was the most active substrate for casein kinase. The results suggest that casein kinase is the enzyme responsible for phosphorylation of many rat liver cytosolic proteins and that several phosphoproteins, apparently under T3-regulation, might be phosphorylated by this enzyme.     

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.     

Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.     

Principal neurofilament-associated protein kinase in squid axoplasm is related to casein kinase I

A cytoskeletal extract of pure axoplasm, highly enriched with neurofilaments (ANF), was prepared from the giant axon of the squid. This ANF preparation also contained potent kinase activities which phosphorylated the Mr greater than 400,000 (high molecular weight) and Mr 220,000 squid neurofilament protein subunits. High salt (1 M) extraction of this ANF preparation solubilized most of the neurofilament proteins and kinase activities and gel filtration on an AcA 44 column separated these two components. The neurofilaments eluted in the void volume of the column while the kinase activities eluted in the 17-44-kDa range of the column. Two major kinase activities were measured in this peak of activity. One of these strongly phosphorylated the phosphate acceptor peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and was completely inhibited by the selective inhibitor of cAMP-dependent kinase Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- NH2 (Wiptide). Since addition of cAMP did not stimulate activity, this suggested that this kinase was a free catalytic subunit of cAMP-dependent kinase associated with the neurofilaments. The second kinase activity most effectively phosphorylated alpha-casein, and this activity was not affected by Wiptide. The alpha-casein phosphorylating activity (ANF kinase) was the principal activity responsible for neurofilament protein phosphorylation, and was not inhibited by various inhibitors against second messenger regulated kinases, suggesting it was related to the casein kinase family. Four lines of evidence indicate ANF kinase was similar to casein kinase I. These were: 1) the apparent molecular weight determined by gel filtration and the chromatographic elution profile on phosphocellulose column corresponded to casein kinase I; 2) heparin, an inhibitor of casein kinase II at 2-5 micrograms/ml, stimulated both ANF kinase and purified casein kinase I at these concentrations, while CKI-7, a relatively selective inhibitor of casein kinase I, inhibited ANF kinase in a comparable dose-response fashion; 3) purified casein kinase I strongly phosphorylated both ANF protein subunits (like ANF kinase) whereas casein kinase II was relatively ineffective; and 4) tryptic peptide maps of the HMW and Mr 220,000 neurofilament proteins after phosphorylation by ANF kinase or purified casein kinase I showed similar 32P-peptide patterns.     

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Summary Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-l-methylxanthine had no effect on NHCP historic kinase activity; the addition of 10 g of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H₂ kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED₅₀ = 0.19 mM) and heparin to inhibit (ID₅₀ = 0.09 g/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activited calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.Abbreviations NHCP Non-Histone Chromatin Proteins - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - PK-C Diacylglycerol-Activated Calcium/phospholipid-dependent Protein Kinase - NK-11 Nuclear Casein Kinase 11 - CK-G Cytosolic Casein Kinase G or 11 - PMSF Phenylmethyl Sulfonyl Fluoride - PKI the Heat Stable PK-A Inhibitor (Walsh inhibitor) - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - EGTA Ethyleneglycol bis- (B-aminoethyl ether) N,N,N,N,-Tetraacetic Acid - PS Phosphatidylserine - DO 1,2-Diolein     

Changes induced by hydrazine in optical spectra of cytochrome oxidase

Casein kinase-TS (Ck-TS), a type-2 casein kinase purified from rat liver cytosol which phosphorylates seryl and threonyl residues N-terminal to acidic clusters, is specifically inhibited by polyglutamyl peptides which are ineffective both on type-1 casein kinase and on cAMP-dependent protein kinase. The inhibition is competitive toward the protein substrate and non-competitive toward ATP. Among the polyglutamates tested (Glu)70 is the most effective (Ki 0.11 microM). (Glu)10 and (Glu)5 are also inhibitors, though less powerful than (Glu)70, while (Glu)3, (Glu)2 and free glutamic acid up to 5 mM are ineffective. These results disclose the possibility that naturally occurring polypeptides containing long stretches of acidic residues may act as physiological inhibitors of type-2 casein kinases.     

The acidic peptide-specific type II protein kinases from the nucleus and the cytosol of porcine liver are distinct

The second messenger-independent acidic peptide-specific protein kinase II (casein kinase II) from the cytosol of porcine liver has been purified to apparent homogeneity by using DEAE-cellulose, hydroxyl apatite, and phosphocellulose chromatography. The native enzyme has an apparent Mr of 150,000. After sodium dodecyl sulfate-gel electrophoresis a band of Mr = 39,000 and a slightly diffuse band of Mr = 27,000 were found indicating an alpha 2 beta 2 structure of this protein kinase. A thorough comparison with the corresponding enzyme from the nucleus was performed. The two enzymes differ in the subunit composition, as the nuclear enzyme is composed of subunits with a Mr of 95,000; they further differ in the heparin sensitivity and binding to blue dextran-Sepharose. Distinct differences in their nucleotide binding sites were found upon mapping with ATP analogs, although both enzymes utilize ATP as well as GTP. On the other hand, both enzymes phosphorylate identical sites in the casein variants beta A2 and alpha S1B at comparable rates. These results demonstrate for the first time the existence of distinct nucleus and cytoplasm specific type II "casein kinases".     

Casein kinase II is a major protein phosphorylating activity in the nuclei of Xenopus laevis oocytes

The nuclei of Xenopus laevis oocytes contain kinases capable of phosphorylating endogenous and exogenous proteins using either ATP or GTP as phosphoryl donors. These enzymes are much more active with casein and phosvitin as substrates than with histones or protamines. The protein phosphorylating activity of oocyte nuclear extracts is not regulated by cyclic nucleotides, phorbol esters, calmodulin and calcium, or phospholipids. However, the casein phosphorylating activity can be greatly enhanced by the polyamines spermine or spermidine and drastically inhibited by heparin. Fractionation of the nuclear casein kinase activities by DEAE-Sephadex chromatography and glycerol gradient centrifugation indicate that the nuclei contain enzymes with the properties of casein kinases I and II as characterized in other species. Oocyte casein kinase I (Mr 37,000) is specific for ATP as phosphoryl donor, is only slightly inhibited by 10 micrograms/ml heparin, and is not significantly stimulated by polyamines. Casein kinase II (Mr 135,000) can use both ATP and GTP as substrates, and is very sensitive to heparin inhibition and polyamine stimulation. The fact that low concentrations of heparin (10 micrograms/ml) can inhibit a large percentage of the endogenous phosphorylation of nuclear extracts or of whole nuclei indicates that casein kinase II is probably the major protein phosphorylating activity of these oocyte organelles.     

Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.     

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.