Rabbit liver glycogen synthase kinases. Characterization of a protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin

A rabbit liver protein kinase (PC0.7), able to phosphorylate glycogen synthase and phosvitin, has been extensively purified. The enzyme had apparent Mr = 170,000-190,000 as judged by gel filtration and was associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000). Two other polypeptides, Mr = 38,000 and Mr = 35,000, were also detected. Treatment with trypsin led to an enzyme composed only of polypeptides of Mr = 35,000 and Mr = 25,000. The beta-polypeptide underwent autophosphorylation when incubated with Mg2+ and ATP or GTP. The protein kinase was effective in utilizing both ATP and GTP as the phosphoryl donor (apparent Km values 5-11 microM and 9-19 microM, respectively). The enzyme phosphorylated phosvitin, casein, and glycogen synthase but not histone or phosphorylase and was inhibited by heparin. Phosphorylation of glycogen synthase proceeded to approximately 0.5 phosphate/subunit with little inactivation of the glycogen synthase. The phosphorylation occurred predominantly in a 21,000-dalton CNBr fragment of glycogen synthase that had been previously shown to reside toward the COOH terminus of the molecule. The liver PC0.7 appeared very similar to an analogous enzyme isolated from rabbit muscle (DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962). The present work, therefore, provides a point of contact between the Ca2+ and cyclic nucleotide-independent glycogen synthase kinases of rabbit liver and muscle.     

Specific phosphorylation of the beta subunit of eIF-2 factor from brain by three different protein kinases

The eukaryotic initiation factor 2 (eIF-2) from calf brain has been purified to homogeneity and free of endogenous kinase activity. Phosphorylation of eIF-2 factor has been examined with four different protein kinases. Casein kinase II, calcium/phospholipid-dependent protein kinase and cyclic AMP-dependent protein kinase from brain, phosphorylate the beta subunit of eIF-2, whilst hemin-controlled inhibitor phosphorylate the alpha subunit of the factor. According to the peptide maps obtained, the phosphorylation sites of the factor by the three beta kinases are specific and distinct. These data suggest a different regulation for the beta subunit through this modification.     

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P-labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB-2 phosphorylation was increased by all three hormones while CB-1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2+-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase II (PC0.7) and FA/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK-3, is involved or phosphatase activity is regulated, or both.     

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.     

The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.     

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.     

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".     

Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit

Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.     

Calmodulin-dependent glycogen synthase kinase: Identification in liver of normal and phosphorylase kinase-deficient rats

We have purified a calmodulin-dependent glycogen synthase kinase from livers of normal and phosphorylase kinase-deficient (gsd/gsd) rats. No differences between normal and gsd/gsd rats were apparent in either (a) the ability of liver extracts to phosphorylate exogenous glycogen synthase in a Ca²⁺- and calmodulin-dependent manner or (b) the purification of the calmodulin-dependent synthase kinase. Although extracts from rat liver, when compared to rabbit liver extracts, had a significantly reduced ability to phosphorylate exogenous synthase, the calmodulin-dependent synthase kinase could be purified from rat liver using a protocol identical to that described for rabbit liver. Moreover, the synthase kinase purified from rat liver had properties very similar to those of the rabbit liver enzyme. The enzyme was completely dependent on calmodulin for activity against glycogen synthase, was unable to phosphorylate phosphorylase b, catalyzed the rapid incorporation of 0.4 mol phosphate/mol of glycogen synthase subunit, selectively phosphorylated sites 1b and 2 in the glycogen synthase molecule, had a Stokes' radius of about 70 Å, and appeared to be composed of subunits of M_r 56,000 and 57,000. These observations led us to conclude that (1) calmodulin-dependent glycogen synthase kinase is distinct from other kinases previously described and (2) the rat liver kinase and the rabbit liver kinase are very similar enzymes.     

Phosphorylation of rabbit liver glycogen synthase by multiple protein kinases

Purified rabbit liver glycogen synthase was found to be a substrate for six different protein kinases: (i) cyclic AMP-dependent protein kinase, (ii) two Ca2+-stimulated protein kinases, phosphorylase kinase (from muscle) and a calmodulin-dependent glycogen synthase kinase, and (iii) three members of a Ca2+ and cyclic nucleotide independent class, PC0.7, FA/GSK-3, and casein kinase-1. Greatest inactivation accompanied phosphorylation by cyclic AMP-dependent protein kinase (to 0.5-0.7 phosphate/subunit, +/- glucose-6-P activity ratio reduced from approximately 1 to 0.6) or FA/GSK-3 (to approximately 1 phosphate/subunit, activity ratio, 0.46). Phosphorylation by the combination FA/GSK-3 plus PC0.7 was synergistic, and more extensive inactivation was achieved. The phosphorylation reactions just described caused significant reductions in the Vmax of the glycogen synthase with little effect on the S0.5 (substrate concentration corresponding to Vmax/2). Phosphorylase kinase achieved a lesser inactivation, to an activity ratio of 0.75 at 0.6 phosphate/subunit. PC0.7 acting alone, casein kinase-1, and the calmodulin-dependent protein kinase did not cause inactivation of liver glycogen synthase with the conditions used. Analysis of CNBr fragments of phosphorylated glycogen synthase indicated that the phosphate was distributed primarily between two polypeptides, with apparent Mr = 12,300 (CB-I) and 16,000-17,000 (CB-II). PC0.7 and casein kinase-1 displayed a decided specificity for CB-II, and the calmodulin-dependent protein kinase was specific for CB-I. The other protein kinases were able, to some extent, to introduce phosphate into both CB-I and CB-II. Studies using limited proteolysis indicated that CB-II was located at a terminal region of the subunit. CB-I contains a minimum of one phosphorylation site and CB-II at least three sites. Liver glycogen synthase is therefore potentially subject to the same type of multisite regulation as skeletal muscle glycogen synthase although the muscle and liver enzymes display significant differences in both structural and kinetic properties.     

The calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Purification, subunit structure and substrate specificity

A calmodulin-dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified approximately equal to 5000-fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0-33%), and chromatographies on phosphocellulose, calmodulin-Sepharose and DEAE-Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of approximately equal to 3%. The Km for glycogen synthase was 3.0 microM and the V 1.6-2.0 mumol min-1 mg-1. The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other. Following incubation with Mg-ATP and Ca2+-calmodulin, the purified protein kinase underwent an 'autophosphorylation reaction'. The reaction reached a plateau when approximately equal to 5 mol of phosphate had been incorporated per 58 000-Mr subunit. Both the 58 000-Mr and 54 000-Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity. The calmodulin-dependent protein kinase initially phosphorylated glycogen synthase at site-2, followed by a slower phosphorylation of site-1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl-CoA carboxylase and ATP-citrate lyase. These findings suggest that the calmodulin-dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo. Glycogen synthase kinase-4 is an enzyme that resembles the calmodulin-dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site-2), but not glycogen phosphorylase. Glycogen synthase kinase-4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin-dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase-4 is not a proteolytic fragment of the calmodulin-dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+-calmodulin.     

Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components

Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S.M. Tahara and J.A. Traugh (1981) J. Biol. Chem. 256, 11558-11564; P.T. Tuazon, W.C. Merrick, and J.A. Traugh (1980) J. Biol. Chem. 255, 10954-10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+-stimulated protease specifically activated protease-activated kinase II; no effect was observed on protease-activated kinase I. The Fe2+-stimulated protease was not active on either protein kinase. The protease-activated kinases were examined using initiation factors (eIF) and 40-S ribosomal subunits as substrate. Protease-activated kinase I phosphorylated one subunit of eIF-3 (Mr 130000), eIF-4B and 40-S ribosomal protein S10. Protease-activated kinase II modified the beta subunit of eIF-2 (Mr 53000) and 40-S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP-dependent and cAMP-independent protein kinases from reticulocytes.     

Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.     

The interferon-induced protein kinase PK-i from mouse L cells.

Interferon-treated L cells are characterized by an increased protein kinase activity that can selectively phosphorylate the small subunit of eukaryotic initiation factor 2. This protein kinase, PK-i, has been extensively purified and shown to be a potent inhibitor of mRNA translation. The purified PK-i contains the endogenously phosphorylated 67,000 Mr protein characteristic of interferon-treated cell extracts. PK-i can also phosphorylate arginine-rich histones. Purified PK-i can be activated by preincubation with ATP (but not adenylyl imidodiphosphate) and low concentrations of double-stranded RNA. The activation results in an increase in the first rate of eIF-2 phosphorylation. Activated PK-i becomes resistant to high concentrations of double-stranded RNA and more thermostable. A stimulator of PK-i activity, factor A, was isolated, as well as a specific phosphoprotein phosphatase that dephosphorylates the 67,000 Mr protein and eIF-2. These two factors, which are present in untreated L cells, may regulate the translation inhibitory activity of the interferon-induced and double-stranded RNA-activated protein kinase PK-i.     

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.     

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.     

A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40–43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent K_m values have been determined to be 15 μM for ATP, 1.2 μM for S6 and 10 μM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n − 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.     

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.