Multiple forms of cytosol and membrane-bound protein kinase activity in human erythrocytes

Both cytosol and membranes of human erythrocytes display protein kinase activity towards exogenous protein substrates such as casein, phosvitin andhistones. The histone kinase activity, unlike casein kinase, of both cytosol and membranes is increased by cyclic AMP. The protein kinase forms removed from the membranes with 0.7 M NaCl, phosphorylate only serine residues of both casein and histones through a mechanism cyclic AMP-independent.The protein kinase activity located in the cytosol (hemolysate) is due also to enzyme forms phosphorylating both serine and threonine residues of casein, in addition to forms phosphorylating only serine residues of casein and histones.Also the cytosol kinase forms, once partially purified by Sepharose 6B filtration, appear to be cyclic AMP-independent.     

Comparative study of mitochondrial and cytosol protein kinase activities.

Both cytosol and mitochondria of rat liver display protein kinase activity, cyclic AMP-independent, which is resolved by Sepharose 6B filtration and P-cellulose chromatography into multiple forms phosphorylating, besides endogenous mitochondrial membrane-bound proteins, also exogenous phosphoproteins such as casein and phosvitin. However, the forms by far predominant in the cytosol phosphorylate both phosphorylserine and phosphorylthreonine residues of casein, while most of the activity associated to mitochondrial structures is due to the forms phosphorylating only phosphorylserine residues.     

Comparative study of mitochondrial and cytosol protein kinase activities

Both cytosol and mitochondria of rat liver display protein kinase activity, cyclic AMP-independent, which is resolved by Sepharose 6B filtration and P-cellulose chromatography into multiple forms phosphorylating, besides endogenous mitochondrial membrane-bound proteins, also exogenous phosphoproteins such as casein and phosvitin.However, the forms by far predominant in the cytosol phosphorylate both phosphorylserine and phosphorylthreonine residues of casein, while most of the activity associated to mitochondrial structures is due to the forms phosphorylating only phosphorylserine residues.     

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.     

Purification of a novel eIF-2 alpha protein kinase from calf brain

A new eukaryotic initiation factor 2 kinase has been purified for the first time from calf brain cytosol. The purification of a nonabundant novel protein kinase activity, designated as PKI, that phosphorylates the alpha subunit of eukaryotic initiation factor 2 is described. The protein kinase activity was assayed using purified initiation factor 2 as a substrate and was purified by ammonium sulphate precipitation, conventional chromatography in heparin-Sepharose and phosphocellulose and by high performance size exclusion and anion exchange chromatographies. The protein kinase activity elutes in the region of 140,000 in the size exclusion chromatography and is associated with two different polypeptides a and b, with relative molecular masses of 38,000 and 20,000 and an approximate ratio of 2.5-3.0:1. The protein kinase does not phosphorylate casein or histones and it is independent of cyclic nucleotides. It can be classified as a serine kinase since the phosphorylation of the alpha subunit of eIF-2 is produced in serine residues. Under these conditions none of the kinase subunits are phosphorylated.     

Activation of a manganese-dependent membrane protein kinase by serine and tyrosine phosphorylation.

A Mn2(+)-dependent serine/threonine protein kinase from rat liver membranes copurifies with the insulin receptor (IR) on wheat germ agglutinin (WGA)-sepharose. The kinase is present in a nonactivated form in membranes but can be activated 20-fold by phosphorylating the WGA-sepharose fraction with casein kinase-1 (CK-1), casein kinase-2 (CK-2), or casein kinase-3 (CK-3). The activated kinase can use IR beta-subunit, myelin basic protein, and histones as substrates. Activation of the kinase seems to proceed by two or more steps. Sodium vanadate and Mn2+ are required in reaction mixtures for activation to be observed, whereas the tyrosine kinase-specific substrate, poly (glu, tyr), completely inhibits activation. These observations suggest that, in addition to serine/threonine phosphorylation by one of the casein kinases, activation of the Mn2(+)-dependent protein kinase also requires tyrosine phosphorylation. Such phosphorylation may be catalyzed by the IR tyrosine kinase.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.     

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.     

Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei.

A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.     

Casein kinase in cytosol of rat and mouse mammary epithelial cells isolated from histone kinase by MgCl2 treatment and influence of pregnancy and lactation on the enzyme activity

Rat mammary glands contain cyclic AMP-independent casein kinase and cyclic AMP-dependent histone kinase. The former was easily isolated from cyclic AMP-dependent histone kinase by MgCl2 treatment. Mammary casein kinase was not activated by cyclic nucleotides, and Mg++ and ATP were required for activation. The specific activity of casein kinase in cytosol of rat mammary epithelial cells increased 2 to 3-fold during pregnancy and lactation. Cytosol of mouse mammary epithelial cells also contained cyclic AMP-independent casein kinase, and the activity of this enzyme was about three times that of the Golgi fraction.     

An ecto-cyclic AMP-independent protein kinase in goat spermatozoa and its change of activity during forward motility

Goat cauda-epididymal intact spermatozoa have been shown to possess an ecto-cyclic AMP-independent protein kinase activity on the external surface that causes phosphorylation of the serine and threonine residues of exogenous phosvitin. The enzyme is neither a tyrosine kinase nor a catalytic subunit of the cyclic AMP-dependent protein kinase. It is not activated by Ca2+, calmodulin and phosphatidylserine. The intact-cell enzyme is capable of phosphorylating a variety of proteins including sperm plasma membrane-bound phosphoprotein(s). The enzymic activity of the intact spermatozoa was not due to contamination of broken or "leaky" cells. The kinase activity of the whole cells was strongly inhibited by the non-penetrating surface probes: p-chloromercuriphenylsulphonic acid (10 microM) and proteases (125 micrograms/ml). The specific activity of the ecto-kinase increased nearly 100% during vigorous forward progression of spermatozoa.     

Endogenous proteinkinase-dependent phosphorylation of rat liver mitochondrial membranes

Isolated rat liver mitochondrial membranes are found to contain tightly bound protein substrate(s) which can be phosphorylated in the presence of ATP by protein kinase(s) previously extracted with 0.7 M NaCl from the membranes themselves and by phosvitin kinase purified from liver cytosol. Such proteinkinase-dependent phosphorylation, which seems to be cyclic AMP-independent, involves the seryl and threonyl residues of the protein substrate(s).     

Identification of multiple protein kinases in normal human lymphocytes.

The protein kinase activity of a 10,000 g supernatant of purified human lymphocytes can be resolved by DEAE-cellulose chromatography into six protein kinase fractions: three of them phosphorylate casein preferentially, and three histones. The same procedure with the corresponding nuclear fraction yields only two casein kinases. All these fractions, except one casein kinase of the cytosol, have been studied with respect to protein and nucleotide specificity, effect of salts and of cyclic nucleotides, sedimentation, etc. The results obtained indicate that the enzyme fractions of the cytosol have distinct characteristics, suggesting that they are different protein kinases, and that the nuclear kinases are similar to the two main casein kinases of the cytosol.     

Adenosine 3′:5′-cyclic phosphate-dependent and -independent protein kinases of renal brush border membranes: Solubilization,separation, and characterization of multiple forms

Multiple protein kinase activities were found in the luminal segment of the renal proximal tubule cell plasma membrane (brush border membrane). Membranes were extracted with Lubrol, with no loss in activity, and the extract was chromatographed on diethylaminoethyl cellulose with a salt gradient. With protamine as substrate, activity eluted in two peaks, designated I and IIb, and was cyclic AMP independent. With histone VII-S, one peak, designated IIa, appeared, which eluted slightly ahead of IIb and was cyclic AMP dependent. The three activities eluted in their original patterns following rechromatography. Histone kinase activity in the combined IIa+b fraction was stimulated threefold by cyclic nucleotides (K_a = 0.013 and 0.94 μM for cyclic AMP and cyclic GMP, respectively) by increasing V. Cyclic AMP binding activity eluted with histone kinase activity. Rechromatography of IIa+b on diethylaminoethyl cellulose containing 1 μm cyclic AMP resulted in passage through the column of most of the histone kinase activity (IIa) prior to the salt gradient, but retention of kinase IIb, which again eluted in its original position. Characterization of the separated enzymes revealed that kinase I was highly specific for protamine and totally insensitive to cyclic AMP and a specific protein inhibitor of cyclic AMP-dependent kinases. Kinase IIa was relatively specific for histones and was completely inhibited by the protein inhibitor. Kinase IIb was nonspecific, catalyzing phosphorylation of protamine, casein, histones, and phosvitin in decreasing order of activity, and was insensitive to cyclic AMP and the protein inhibitor. Exposure of intact brush border membranes to elevated temperatures revealed that phosphorylation of intrinsic membrane proteins and protamine was thermolabile, whereas cyclic AMP-dependent histone kinase activity was relatively thermostable. These findings implicate cyclic AMP-independent protamine kinases in the cyclic AMP-independent autophosphorylation of the brush border membrane.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts.

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743–745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L₆ myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system.We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f₂b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10^?5 M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase.Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems.The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane missing data     

Phosphorylation of hormone-sensitive lipase by cyclic GMP-dependent protein kinase

In intact rat adipocytes hormone-sensitive lipase has been shown to be phosphorylated on serine residues in two different phosphorylation sites: a regulatory site phosphorylated by cyclic AMP-dependent protein kinase and a basal site, which does not directly affect the enzyme activity, phosphorylated by cyclic AMP-independent protein kinase(s) [(1984) Proc. Natl. Acad. Sci USA 81, 3317-3321]. Cyclic GMP-dependent protein kinase catalyzed the phosphorylation of the same two phosphorylation sites on the isolated enzyme, at serine residues. Both sites were phosphorylated at about the same rate, with the hormone-sensitive lipase activity concomitantly enhanced.     

Subcellular alterations in cyclic AMP-binding and protein kinase activities during ontogeny in the rat cerebellum

Ontogenic relationships between levels of cyclic AMP-binding activity and protein kinase activity were examined in subcellular fractions of the cerebellum during the first 3 weeks of neonatal life. A progressive increase in cyclic AMP levels was paralleled by an increase in cyclic AMP bindign by the nuclear and cytosol fractions, but not by the mitochondrial or microsomal fractions. Utilization of heat-stable protein kinase inhibitor permtited distinction of the cyclic AMP-dependent from the cyclic AMP-independent form of the protein kinase population. Cyclic AMP-dependent protein kinase increased between days 4 and 20 to represent a progressively greater proportion of the protein kinase population. In all subcellular fractions alterations of cyclic AMP-dependent protein kinase during neonatal development paralleled changes in binding of cyclic AMP to protein in these fractions. In both the nuclear and cytosol fractions cyclic AMP-dependent protein kinase activity increased progressively between days 4 and 20, i.e. 64 ± 6 to 176 ± 16 and 79 ± 12 to 340 ± 12 pmol/min per mg protein, respectively. Cyclic AMP-dependent protein kinase activity in the mitochondrial fraction declined during the postnatal period studied, and in the microsomal fraction it rose to a non-sustained peak at 14 days and fell thereafter. Unlike the cyclic AMP-dependent form, cyclic AMP-independent protein kinase activity did not follow the ontogenetic pattern of cyclic AMP-binding activity. The specific activity of nuclear cyclic AMP-independent protein kinase did not change during days 4–20, and a non-sustained rise of cyclic AMP-independent protein kinase activity in both cytosol and microsomal fractions during the 7th–12th day tended to parallel more closely known patterns of postnatal proliferative growth. The findings reported herein indicate that the ontogenic pattern of cyclic AMP-dependent protein kinase varies between different subcellular fractions of the neonatal cerebellum, that these patterns parallel the changes in cyclic AMP-bidign activity, and suggest that the component parts of the cyclic AMP system may develop as a functional unit.     

A type-1 casein kinase from yeast phosphorylates both serine and threonine residues of casein. Identification of the phosphorylation sites

A protein kinase (casein kinase 1A) active on casein and phosvitin but not on histones has been purified to near homogeneity from yeast cytosol and meets most criteria for being considered a type-1 casein kinase: it is a monomeric enzyme exhibiting an Mr of about 27 kDa by sucrose gradient centrifugation: it is not affected by inhibitors of type-2 casein kinases, such as heparin and polyglutamate, and shows negligible affinity for GTP. It also readily phosphorylates the residue Ser-22 of beta-casein located within the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser22-Ile-Thr-Arg- which is typically affected by casein kinases of the first class. On the other hand, casein kinase 1A displays the unusual property of phosphorylating threonine residue(s) in both whole casein and alpha s1-casein. The threonine residue phosphorylated in alpha s1-casein and accounting for most of the 32P incorporated into this protein by casein kinase 1A has been identified as Thr-49, which occurs in the sequence -Ser(P)-Glu-Ser(P)-Thr(P*)49-Glu-Asp-Gln-, whose two Ser(P) residues are already phosphorylated in the native protein. It is concluded that some type-1 casein kinases can also phosphorylate threonine residues provided they fulfil definite structural requirements, probably an acidic cluster near their N-terminal side.