Purification of rat liver nuclear protein kinase NI.

Rat liver nuclear protein kinase NI, which appears in the flowthrough of DEAE-Sephadex columns, has been purified approximately 15,000-fold from soluble nuclear protein with yields of up to 10%. The method of purification involved chromatography of the DEAE-flowthrough protein successively on phosvitin-Sepharose and casein-Sepharose followed by rechromatography on phosvitin-Sepharose. The purified enzyme has an s20,w and molecular weight of 3.7 and 47,000, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. A similar molecular weight of 42,000 was determined by gel filtration using Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide with a molecular weight of 25,000. Protein kinase NI therefore consists of a dimer of two identical subunits. Protein kinase NI exhibits maximal activity on casein substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP when either casein or histone H2b is used as a substrate.     

Occurrence of protein kinases NI and NII in human and porcine thyroids

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were detected in the nuclei of human and porcine thyroid tissues, and compared with those from rat liver. Enzymes were extracted from the isolated nuclei with a buffer solution containing 0.4 M NaCl, and analyzed by DEAE-Sephadex and phosphocellulose column chromatographies. The chromatographies, together with the characterization of the enzymes, demonstrated that human and porcine thyroid tissues contained two major casein kinases in the cell nuclei, the properties of which revealed that they are to be identified as protein kinases NI and NII.     

Rat liver nuclerar protein kinases.

We have shown that nuclei isolated by two methods contain grossly different amounts of cyclic AMP-dependent histone kinase activity. Repeated washing of the isolated nuclei with a low ionic strength buffer removed the majority of the cyclic AMP-dependent histone kinase and cyclic AMP binding activity. Nuclear cyclic AMP-dependent histone kinase activity accounted for only 0.42% of the total cytoplasmic enzyme activity. Similarly, the lactate dehydrogenase activity associated with liver nuclei represented only 0.07% of the total cytoplasmic activity. The isolated liver nuclei contained only 0.27% of the total homogenate glutamate dehydrogenase activity and 1.7%of the total homogenate glucose-6-phosphatase activity. The cyclic AMP-dependent histone kinase behaves as a cytoplasmic rather than a nuclear enzyme. We have also shown that using crude extracts, one can achieve separation of the two nuclear casein kinases, NI and NII, on sucrose density gradients in the presence of 0.5M NaCl. Nuclear casein kinases NI and NII had sedimentation coefficients of 3.0 and 593 S, respectively, in the presence of 0.5 M NaCl. Under conditions of low ionic strength, all of the casein kinase activity in the crude nuclear extract sedimented as one peak with a seminentation coefficient of 7.3 S. The aggregation-disaggregation which occurred in the crude extract was reversible and was mainly due to the aggregative and disaggregative properties of casein kinase NII. The two nuclear casein kinases have different affinities for chromatin. When nuclei were disrupted in a hypotonic solution and extracted with a buffercontaining 0.14 M NaCl, casein kinase NII could be completely extracted from the viscous nuclear material. Although a significant amount of casein kinase NI was extracted by the buffer containing 0.14 M NaCl, re-extraction of the nuclear material with a buffer containing 0.5 M NaCl yielded substantial amounts of casein kinase NI, and a final extraction with a buffer containing 1.0 M NaCl yielded measurable amounts of casein kinase NI. No casein kinase NII activity could be detected in the 0.5 M and 1.0M NaCl extracts.     

Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver

Damage to DNA can result in strand breaks with 5′-hydroxyl and 3′-phosphate termini. Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 5′-phosphate and 3′-hydroxyl groups. Polydeoxynucleotide kinase is an enzyme that may fulfil this function. We have purified the kinases from calf thymus and rat liver to near homogeneity. Based on SDS-polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are ∼60-kDa polypeptides. Both enzymes have an acidic pH optimum (5.5–6.0) for kinase activity, and similar pl values (8.5–8.6), and a specificity for DNA. The calf thymus kinase possesses a 3′-phosphatase activity, as has previously been shown for the rat liver enzyme. The minimum size of oligonucleotide that can be labelled is 7–8 nucleotides in length, but the optimal size appears to be >18 nucleotides. Comparison of phosphorylation of oligo(dA)₂₄ and oligo(dT)₂₄ with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates. Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 5′-overhanging termini when acting at DNA termini produced by restriction enzymes. With double-stranded oligonucleotide complexes designed to model single-strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 5′-terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single-strand breaks. J. Cell. Biochem. 64:258–272. © 1997 Wiley-Liss, Inc.     

Separation of nuclear cAMP independent protein kinases NI and NII from their chromosomal protein substrates and enzyme inhibitors by the use of a casein-phosvitin-Sepharose column

A casein-phosvitin-Sepharose chromatography column allows separation of nuclear protein kinases from their chromosomal phosphoprotein substrates and from at least some protein kinase inhibitors in a single step. The additional step of passing the eluted material through a partially hydrolyzed, dephosphorylated casein-Sepharose column separates the two protein kinases, NI and NII, from each other.     

Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.     

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.     

Rat liver polysome N alpha-acetyltransferase: substrate specificity.

The substrate specificity of polysome rat liver N alpha-acetyltransferase (NAT) has been examined by utilizing a series of synthetic and natural substrates that has been systematically altered with respect to N-terminal sequence and length. Families of peptides of the structure S-Y-S-G-G-L-L-L were generated by successively replacing the N-terminal serine, the penultimate tyrosine, and the antepenultimate serine with all 19 commonly occurring amino acids, which were then assessed for their reactivity with the rat liver enzyme. Only peptides with N-terminal serine, alanine, methionine, leucine, and phenylalanine were modified. Glycine, lysine, arginine, valine, isoleucine, and tryptophan in the second position are (with N-terminal serine) strongly inhibitory, and proline completely blocks modification. Third-position substitutions have less of an effect on NAT activity with glycine, aspartic acid, glutamic acid, and tryptophan being most inhibiting (with N-terminal Ser-Tyr). These observations are generally in agreement with in situ modifications although there are some significant differences particularly with respect to the amino-terminal residues. Optimal chain length was determined to be 10-11 residues with either synthetic peptides of the structure S-Y-S-(G)n-L-L-L or adrenocorticotropin (ACTH) sequences ranging from 8 to 39 residues. The ACTH peptides were generally found to be severalfold better substrates than the corresponding synthetic ones. Activity was not affected by increased chain length beyond approximately 17 residues. These data support the view that polysome-catalyzed N alpha-acetylation occurs as a cotranslational event on nascent chains of about 20-40 amino acids in length.     

In vitro substrate specificity of protein tyrosine kinases

Synthetic peptides such as P60^stc autophosphorylation site peptides and angiotensin are indiscriminately phosphorylated by protein tyrosine kinases. The observation has led to the general belief that protein tyrosine kinases are highly promiscuous, displaying littlein vitro site specificity. In recent years, evidence has been accumulating to indicate that such a belief requires close examination. Synthetic peptides showing high substrate activity for specific groups of protein tyrosine kinases have been obtained. Systematic modification of certain substrate peptides suggests that kinase substrate determinants reside with specific amino acid residues proximal to the target tyrosine. A number of protein kinases have been shown to be regulated by tyrosine phosphorylation at specific sites by highly specific protein tyrosine kinases. These and other selected biochemical studies that contribute to the evolving view ofin vitro substrate specificity of protein tyrosine kinases are reviewed.     

Rat adrenocortical carcinoma 494 autophosphorylating protein kinase, autophosphorylating protein kinase 500. Purification, biochemical and immunological characterization, and substrate specificity

A novel autophosphorylating protein kinase, autophosphorylating protein kinase 500, independent of cyclic AMP, cyclic GMP, calcium, and calmodulin was purified from rat adrenocortical carcinoma 494 by ammonium sulfate fractionation followed by the chromatographic steps of DEAE-cellulose, gel filtration, cyclic AMP-epoxy Sepharose, and phosphocellulose. Sometimes two additional chromatographic purification steps of chromatofocusing and gel filtration were necessary for complete purification. The enzyme was homogeneous as evidenced by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sucrose density sedimentation studies indicated that Mr of the enzyme was 490,000, while ultracentrifugal analysis demonstrated a value of 481,400 (+/-7%). The protein was composed of two identical subunits each with Mr = 250,000. The enzyme molecule was slightly asymmetric with frictional and sedimentation coefficients of 1.28 and 18.20, respectively, and a Stokes radius of 66 A. Isoelectric focusing electrophoresis revealed a single peak with pI 4.6, indicating acidity of the protein. The enzyme self phosphorylated one or more of its serine residues. The reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mM Mn2+ or 10 mM Mg2+) were essential for optimum activity. Autophosphorylating protein kinase 500 did not phosphorylate the commonly used exogenous substrates such as histones, casein, phosvitin, or protamine. Analysis of autophosphorylating protein kinase 500 with rabbit anti-autophosphorylating protein kinase 500 IgG by immunoelectrophoresis and crossed immune electrophoresis demonstrated single arcs of precipitation, confirming the biochemical demonstration of enzyme purification and homogeneity. Indirect immunofluorescence studies revealed an intracytoplasmic localization of the enzyme in cultured and freshly isolated adrenocortical carcinoma 494 cells. Both cell types revealed an intensity of perinuclear enzyme fluorescence, but an absence of the enzyme in the nuclei or nucleoli. The anti-autophosphorylating protein kinase 500 IgG blocked the self-catalyzed phosphorylation of autophosphorylating protein kinase 500, providing immunological support of the biochemical results that autophosphorylation is an intrinsic characteristic of the enzyme. When autophosphorylating protein kinase 500 was incubated with membrane-bound ribosomes, it phosphorylated a Mr = 31,000 protein. This phosphorylation was blocked by the anti-autophosphorylating protein kinase 500 IgG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of rat liver nuclear protein kinases.

Two cyclic nucleotide-independent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) have been purified to homogeneity from rat liver nuclei. While these enzymes have many similar catalytic properties (preference for acid rather than basic proteins), they differ in molecular weight and subunit composition. Protein kinase NII will utilize ATP and GTP as phosphate donors while protein kinase NI will only effectively use ATP. Both enzymes reveal an unusual activation by Fe2+.     

The separation, properties and possible subunit composition of adenosine 3',5'-monophosphate-dependent protein kinases in brown adipose tissue.

Two 8.5-S protein kinases (ATP : protein phosphotransferase EC 2.7.1.37) and one 6.6-S protein kinase were purified 500--1000-fold from the acid-soluble fraction of brown adipose tissue. The catalytic properties of the kinases were similar. Each kinase was activated by cyclic AMP and had two components of cyclic AMP binding. In the presence of 200 nM cyclic AMP, undissociated kinase activity sedimented at 7.7 or 5.5 S. Free catalytic activity (3.2 S) could be detected but was unstable. Free regulatory units could not be detected. The 8.5-S protein kinase was dissociated by freezing and thawing to a 7.7-S variety with loss of the higher affinity component of binding. The 7.7-S kinase was sedimented through linear gradients of sucrose containing different concentrations of cyclic AMP. At each concentration, kinase activity lost from the holoenzyme peak (% of original) was identical with the amount of cyclic AMP bound at equilibrium (% oof maximum). Similar experiments on the 8.5-S kinase showed that the binding component with higher affinity was not associated with the release of catalytic activity. The results were consistent with the propostal that the kinases isolated contained one more cyclic AMP binding subunit than catalytic subunit (3 : 2 for 8.5 S and 2 : 1 for 6.6 S) and that this extra subunit was released to give an equal number of subunits of each type before catalytic activity was liberated.     

Molecular basis for substrate specificity of protein kinases and phosphatases

Regulation of various metabolic processes occurs by the phosphorylation/dephosphorylation of enzymes. Both the protein kinases that catalyze the phosphorylations and the protein phosphatases that catalyze the dephosphorylations display relatively broad specificity, reacting with a number of distinct sites in target enzymes. In this way changes in the activity of a particular kinase or phosphatase can cause coordinated and pleiotropic responses. However, the kinases and phosphatases do not exhibit a one-to-one correspondence in their reactions. Residues at different positions may be phosphorylated by a single kinase, yet dephosphorylated by different individual phosphatases. Conversely, sites which are substrates for different individual kinases may be dephosphorylated by a single phosphatase. In exploring the molecular basis for these differences this article shows that whereas kinases react with specific primary structures that often times appear as beta bends, the phosphatases recognize higher order structure, less strictly ruled by amino acid sequence surrounding the phosphorylated site. The differences, seen in the ability of these enzymes to utilize synthetic peptide substrates, might be rationalized in terms of function. Kinases need protruding segments of structure that can be enwrapped to exclude water, thereby minimizing ATP hydrolysis and enhancing phosphotransferase activity. On the other hand phosphatases are hydrolytic enzymes that may operate especially well on protein interfaces. Hydrolytic action often measured with p-nitrophenylphosphate is not necessarily indicative of a protein phosphatase and consideration of the mechanism reveals why this substrate can be misleading.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Rat epididymal alpha-D-mannosidase: purification, carbohydrate composition, substrate specificity, and antibody production

Two alpha-D-mannosidases have previously been identified in rat epididymis. This communication reports the purification and characterization of the "acid" alpha-D-mannosidase. The enzyme was purified over 1000-fold to near homogeneity by acetone and (NH4)2SO4 precipitation followed by ion-exchange and hydroxylapatite chromatography. The molecular weight of the enzyme was estimated to be 220,000 by gel filtration. Polyacrylamide gel electrophoresis of the native enzyme under two conditions of buffer and pH showed a single band when stained for protein while electrophoresis under denaturing conditions resulted in bands of apparent Mr 60,000 and 31,000. The enzyme is a glycoprotein containing about 5.6% hexose. In addition to mannose (3.1%) and glucosamine (2.0%), the enzyme also contained small amounts of glucose, fucose, and galactose. Chemical analysis indicated the absence of sialic acid. The substrate specificity of the purified enzyme was investigated using linear and branched mannose-containing oligosaccharides. The enzyme cleaved linear oligosaccharides [Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc and Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc] very efficiently. However, little or no activity was observed toward high mannose oligosaccharides (Man9GlcNAc through Man5GlcNAc) or the branched trimannosyl derivative Man3GlcNAc. This specificity is very similar to that observed with rat kidney lysosomal alpha-D-mannosidase. Additional evidence that the epididymal enzyme is essentially a lysosomal alpha-D-mannosidase is the fact that polyclonal antibody prepared against the purified epididymal enzyme cross-reacted with lysosomal alpha-D-mannosidase from several rat tissues and with acidic alpha-D-mannosidase of a human cell line, results suggesting that the antibody will be useful in studying the biosynthesis and turnover of lysosomal alpha-D-mannosidases in at least two species.     

Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.     

Purification and partial characterization of the catalytic subunit of cAMP-dependent protein kinases from reticulocytes.

The catalytic subunit of cyclic AMP-dependent protein kinases from rabbit reticulocytes has been purified to near homogeneity. It has a molecular weight of 43,000 as judged from gel filtration and by polyacrylamide gel electrophoresis in the presence of sodium dodecyi sulfate and appears to be similar in physical properties and substrate specificity to the comparable enzyme isolated from muscle or liver. The enzyme phosphorylates histones, a protein of 40 S ribosomal subunits from reticulocytes and from Artemia salina, and the low molecular weight heat-stable phosphatase inhibitor (G. A. Nimmo and P. Cohen, 1978, Eur. J, Biochem.87, 341–351). No evidence has been obtained for a direct or indirect role of this enzyme in the regulation of protein synthesis.