Protein Kinase NII Research Articles

Protein kinase NII. Interaction with RNA polymerase II and contribution to immunological cross-reactivity of RNA polymerases I and II

The interaction between antibodies directed against RNA polymerase I purified from Morris hepatoma 3924A and homologous RNA polymerase II was investigated. The activity of partially purified polymerase II was inhibited by the antibodies. In contrast, the reaction catalyzed by the purified enzyme was not affected. Partially purified polymerase II preparations contained a protein kinase activity. Sucrose gradient centrifugation in the presence of 0.3 M KCl resulted in complete separation of RNA polymerase II from protein kinase as well as in complete loss of sensitivity to the anti-RNA polymerase I antibodies. The protein kinase possessed reaction characteristics similar to those of the NII protein kinase (Rose, K.M., Bell, L.E., Siefken, D.A. and Jacob, S.T. (1981) J. Biol. Chem. 256, 7468–7477) which is associated with hepatoma RNA polymerase I (Rose, K.M., Stetler, D.A. and Jacob, S.T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2833–2837). The activities of both kinases were inhibited to the same extent by anti-RNA polymerase I antibodies and polypeptides of M r 42000 and 25000, present in both kinase preparations, formed immune complexes with the antisera. Readdition of protein kinase NII to purified polymerase II resulted in phosphorylation of the polymerase and a concomitant enhancement of RNA synthesis. After addition of the kinase, RNA polymerase II activity was again sensitive to anti-RNA polymerase I antibodies. Upon reacting with protein kinase NII, RNA polymerase II polypeptides could be detected in immune complexes with anti-RNA polymerase I antibodies. These data indicate that protein kinase NII is associated with RNA polymerase II during early stages of purification and is at least partially responsible for the immunological cross-reactivity of RNA polymerases I and II.

Phosphorylation of high mobility group proteins 14 and 17 by nuclear protein kinase NII in rat C6 glioma cells

High mobility group (HMG) proteins 14 and 17 of rat C 6 glioma cells are phosphorylated in vivo on both serine and threonine. In HMG 14 about 60% of the total [ 32P]phosphate was identified as phosphoserine and 40% as phosphothreonine. In HMG 17, there was 88% phosphoserine and 12% phosphothreonine. Glioma cell nuclear protein kinase NII phosphorylates HMG 14 and 17 in vitro on serine as well as threonine and the relative percentages of [ 32P]phosphoamino acid are similar to those seen in vivo . Nuclear protein kinase NI and the type I and II cAMP-dependent protein kinases exhibit only minor phosphorylating activity towards HMG 14 and 17. We conclude that nuclear protein kinase NII is responsible for the phosphorylation of HMG 14 and 17 in vivo .

Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis.

RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.

Polyamines alter the substrate preference of nuclear protein kinase NII.

When investigating the effects of polyamines on substrate specificity of adenosine cyclic 3', 5'-monophosphate (cAMP) independent nuclear protein kinase NII, we found that the substrate preference of NII was drastically altered in the presence of polyamines. When casein was used as a substrate, spermine stimulated the enzyme activity 7.3-fold at 2 mM, and a 6.5-fold stimulation was observed with 6 mM spermidine. Putrescine was also slightly effective at higher concentrations. With phosvitin as the substrate, however, 2 mM spermine and 4 mM spermidine strongly inhibited the activity by 93% and 80%, respectively, while putrescine showed a weak stimulatory effect. Steady-state kinetics and binding studies suggested that both stimulatory and inhibitory effects of polyamines on NII enzyme activity are probably due to substrate protein-polyamine interactions. The circular dichroism spectrum of phosvitin was apparently altered by spermine, whereas no significant change was observed with casein.

Substrate specificity of the nuclear protein kinase NII from porcine liver. Studies with casein variants.

The substrate specificity of the nuclear protein kinase NII from pig liver was studied using purified casein variants alpha s1B and beta A2 both in the phosphorylated and dephosphorylated form. Chymotryptic peptides of the phosphorylated casein were analyzed by ascending thin-layer chromatography followed by thin-layer electrophoresis. Phosphorylated peptides were detected by autoradiography. The determination of a phosphorylated site in a cluster of three serinees was performed by solid phase Edman degradation of the whole beta A2 casein. Phosphorylated beta A2 casein was by far the best substrate. A threonine at position 41 in the amino acid sequence was found to be predominantly phosphorylated. Dephosphorylated beta A2 casein became rephosphorylated at serine-17 whereas phosphorylated at threonine-41 was greatly reduced. In both sites acidic amino acid side chains are present at the C-terminal side though at different positions suggesting that acidic residues in the vicinity of the serin or threonine residue to be phosphorylated play an important role in the recognition by the nuclear protein kinase NII.

Reversal of heparin inhibition of nuclear protein kinase NII by polyamines and histones

FEBS LettersVolume 128, Issue 1 p. 33-36 Full-length articleFree Access Reversal of heparin inhibition of nuclear protein kinase NII by polyamines and histones Toshiro Hara, Toshiro Hara Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, JapanSearch for more papers by this authorKeikichi Takahashi, Keikichi Takahashi Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, JapanSearch for more papers by this authorHideya Endo, Hideya Endo Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, JapanSearch for more papers by this author Toshiro Hara, Toshiro Hara Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, JapanSearch for more papers by this authorKeikichi Takahashi, Keikichi Takahashi Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, JapanSearch for more papers by this authorHideya Endo, Hideya Endo Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, JapanSearch for more papers by this author First published: June 01, 1981 https://doi.org/10.1016/0014-5793(81)81072-1Citations: 34AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume128, Issue1June 01, 1981Pages 33-36 ReferencesRelatedInformation

Phosphorylation of HMG 17 by protein kinase NII from rat liver cell nuclei

Phosphorylation of HMG 17 by protein kinase NII from rat liver cell nuclei

Preferential phosphorylation of basic non-histone proteins by nuclear protein kinase NII from rat liver.

Rat liver nuclear protein kinase NII, which is independent of cyclic nucleotides, phosphorylated both the acidic protein casein and the basic histone preparation, soluble in 0.25 M HCl, which was extracted from the cell nuclei. When the isolated nuclei were incubated with [32P]ATP in the presence of protein kinase NII, more than 95% of the 32P-labelled proteins was recovered in the 0.25 M HCl extract. In order to analyze the substrate proteins of protein kinase NII, a histone preparation free from the endogenous protein kinase activities was used as substrate. The results demonstrated that histones were not phosphorylated, except for a faint 32P incorporation into the H3 fraction. Instead, multiple non-histone proteins were highly phosphorylated, which were present in a minor quantity in the histone preparation. The molecular weights of the main phosphorylated proteins were 72000, 68000, 56000, 47000, 46000, 43000, 38000 and 32000. Isoelectric focusing demonstrated the basic nature of the phosphorylated proteins, and as much as 72% of 32P radioactivity was distributed in the pH region higher than 7.0. Furthermore, many proteins phosphorylated in the isolated nuclei with added protein kinase NII were also found to be basic non-histone proteins. These results indicate that protein kinase NII preferentially phosphorylates in vitro a set of nuclear basic non-histone proteins.

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 acidic rather than basic proteins), the 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 Fe 2+.

Purification of rat liver nuclear protein kinase NII.

Purification of rat liver nuclear protein kinase NII.

Purification and properties of rat liver nuclear protein kinases.

Two protein kinases, designated NI and NII, have been isolated from rat liver nuclei. These enzymes have a similar pH optimum and phosphorylate phosvitin and casein more readily than histone. Both enzymes require magnesium for activity. In the absence of Mg2+, other divalent cations such as Ca2+, Co2+, and Mn2+ can substitute partially for Mg2+ when the reaction is catalyzed by NI. With NII, only Co2+ showed any activity in the absence of Mg2+. Magnesium decreased the apparent Km for ATP of protein kinase NI without changing the Vmax of the reaction, and decreased the apparent Km's for both ATP and casein, while increasing the Vmax of the reaction threefold with protein kinase NII. Both enzymes are stimulated about twofold by low concentrations (0.1–0.3 M) of NaCl, KCl, and sodium acetate, whereas higher concentrations (> 0.5 M) inhibit their activities. Both enzymes are inhibited by low concentrations of NaF (0.02 M) and (NH4)2SO4 (0.1 M). NI and NII were found to have sedimentation coefficients of 3.6 S and 10.8 S, respectively. The nuclear protein kinases are not activated by cyclic AMP or cyclic GMP, and are not inhibited by the heat-stable cyclic AMP-dependent protein kinase inhibitor.