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

Inoue, Akira; Tei, Yoko; Hasuma, Tadayoshi; Yukioka, Munehiko; Morisawa, Seiji

doi:10.1016/0014-5793(80)80915-x

Cited by 43 publications

(12 citation statements)

References 18 publications

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“…Histones and HMG proteins were isolated from rat liver nuclei, as previously described in [I21 and [8], respectively. 40s ribonucleoprotein (RNP) particles, DNA and nuclear RNA were prepared from the isolated rat liver nuclei as described in [13], [I41 and [15], respectively.…”

Section: Preparative Methodsmentioning

confidence: 99%

“…Rat liver protein kinase NII was purified from a 0.4 M NaCl nuclear extract by three successive column chromatographies (DEAE-Sephadex, Bio-Gel A-3.5 m and phosphocellulose) as previously described in [8], and finally by heparinSepharose chromatography'. The enzyme was more than 90 "/, pure, as determined by SDS gel electrophoresis.…”

Section: Purification Of Protein Kinuse Niimentioning

confidence: 99%

“…Rat liver nuclei were isolated through 9 tissue volumes (v/w) of 2.3 M sucrose, 3 mM MgC12, 0.2 mM phenylmethylsulfonyl fluoride, as described previously [8], and washed with 15 tissue volumes of 0.3 M sucrose, 3 m M MgCI2, 0.2 m M phenylmethylsulfonyl fluoride, 12 mM Tris/HCl, pH 7.6, by gentle stirring at 0 "C for 20 min followed by centrifugation at 3000 x g. The two supernatants (NS1 and NS2) were saved, and the pelleted nuclei, suspended in the same solution containing 0.5 m M CaClz at 30 AZbo units/ml, were incubated with DNase I (2.0-2.5 pg/ml, Sigma, DN-CL) or niicrococcal nuclease (25 units/ml, Worthington) at 30°C for a determined time. At the end of incubation the mixture was chilled on ice, EDTA added to make a final concentration of 5 mM (with 0.1 M solution), and the preparation centrifuged at 3000 x g for 10 min.…”

Section: Preparation Of Si Proteinsmentioning

confidence: 99%

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A set of non‐histone proteins isolated from the nuclei of various rat tissues

Inoue

Higashi

Hasuma

et al. 1983

European Journal of Biochemistry

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A set of non-histone proteins has been identified in the nuclei from liver, brain, spleen and testis tissues of the rat. Following moderate digestion of thoroughly washed nuclei with DNase 1 or micrococcal nuclease, EDTA was added to 5 m M to the reaction mixture and the preparation centrifuged. We found that the supernatant contained a limited amount of non-histone proteins (fraction SI). Sodium dodecyl sulfate (SDS) gel electrophoresis revealed S1 to be composed of a remarkably simple set of proteins resolved into four groups (A -D) each possessing closely spaced doublets or a triplet. Their molecular weights were A, 76100-80000; B, 48200-49500; C, 44500-45200 and D, 39500-41 500. The yield suggested that these proteins were structural constituents; however, they did not coincide with the known structural proteins of the cell nucleus. Two-dimensional gel electrophoresis further resolved each of the SDS bands into as many as nine spots, according to various charges. Some were labelled with [32P]orthophosphate in vivo, or with [y-32P]ATP and purified nuclear protein kinase NII in vitro. The released proteins B -D had fairly constant relative molar ratios at various times of digestion, thereby indicating possible localizations a t similar sites in the nucleus. The kinetic data together with the aggregation property at neutral pH values and the solubility in 5 mM EDTA suggest that proteins B -D constitute a group of proteins that have several common characteristics.The abundance of protein species in the cell nucleus varies with the protein; some are structural or regulatory in function while others are catalytic in various biochemical processes. Isolation and characterization of these nuclear proteins should greatly aid in understanding the structure and function of the nucleus as well as the molecular properties of the protein in question. Isolation of a group of proteins which share molecular properties may be advantageous, as a common biological function may be involved. The precedent of this can be illustrated with histones and high mobility group (HMG) proteins. Histones, the building molecules of the nucleosomes [I], can be readily extracted in a set from the chromatin with dilute mineral acids [2]; H M G proteins, which confer a structural characteristic of the active Chromatin [3], are extracted as a group from chromatin with 0.35 M NaCl and remain soluble in 2There have been numerous studies on various nuclear architectures such as chromatin, nuclear matrix and nuclear envelopes. One approach is to use DNA-hydrolyzing enzymes ; for example, DNase I has been used to distinguish the transcriptionally active from the bulk chromatin in differential digestion kinetics [5], or to isolate the nuclear pore complexlamina fraction [6] and nuclear matrix [7].In our studies on nuclear proteins we were interested in protein components liberated from the nuclei by digestion with DNA-hydrolyzing enzymes. We report here that a novel and remarkably simple set of non-histone proteins was isolated from the nuclei by nucle...

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Section: Preparative Methodsmentioning

confidence: 99%

Section: Purification Of Protein Kinuse Niimentioning

confidence: 99%

Section: Preparation Of Si Proteinsmentioning

confidence: 99%

See 1 more Smart Citation

A set of non‐histone proteins isolated from the nuclei of various rat tissues

Inoue

Higashi

Hasuma

et al. 1983

European Journal of Biochemistry

Self Cite

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“…Protein phosphorylation has been implicated in cell growth control [2] and cell cycle regulation [3] and reported to regulate the action of many proteins that are involved in DNA and RNA metabolism. Among these are RNA polymerases I and I1 [4-61, DNA topoisomerases type I [7] and type I1 [8,91, poly(A) polymerase [lo], nucleolar protein B23 [ll], the highmobility-group proteins HMG14 [12] and HMG17 [13] and reverse transcriptase from Rous sarcoma virus [14].…”

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confidence: 99%

Casein kinase II phosphorylates DNA‐polymerase‐α–DNA‐primase without affecting its basic enzymic properties

Podust¹,

Bialek

Sternbach

et al. 1990

European Journal of Biochemistry

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Immunoaffinity-purified DNA-polymerase-a -DNA-primase complex from calf thymus was phosphorylated in vitro by highly purified casein kinase I1 from the same tissue. Specific phosphorylation of the DNA-polymerizing a subunit and the primase-associated y subunit was observed. About 1 mol phosphate/mol polymerase -primase was incorporated. Despite this effect, neither the DNA polymerase nor the DNA primase activity were changed after phosphorylation by casein kinase 11. Furthermore, dephosphorylation of polymerase -primase with alkaline phosphatase did not change the polymerase or the primase activity to a significant extent. Moreover, both alkaline phosphatase and casein kinase I1 had no effect on the processivity of DNA synthesis and on the lengths and amounts of primers formed by the DNA primase. Because DNA polymerase a maintained all its basic properties even after extensive treatment with alkaline phosphatase, it is unlikely that phosphorylation has a direct influence on the activities of the DNA-polymerase-a -DNA-primase complex. The possible influence of post-translational phosphorylation on the formation of a complex of polymerase a and its accessory proteins is discussed.Phosphorylation and dephosphorylation of proteins modulates the activities of many enzymes in eukaryotic cells [l]. Protein phosphorylation has been implicated in cell growth control [2] The essential DNA-synthesizing complex of eukaryotic cells, DNA-polymerase-a -DNA-primase, has been demonstrated to be a phosphoprotein in vivo [15 -171. Furthermore, phosphorylation and dephosphorylation of crude or partially purified fractions of DNA polymerase a were associated with activation and inactivation events, respectively [16, 18 -211. Therefore, phosphorylation/dephosphorylation might be an important contributor for the cellular regulation of this enzyme and, possibly, for the onset of DNA synthesis in vivo.In this study, we have investigated the influence of in vitro phosphorylation and dephosphorylation on the DNApolymerizing and RNA-priming capabilities of the immunoaffinity-purified DNA-polymerase-a -DNA-primase complex from calf thymus. We demonstrate that highly purified casein kinase I1 (CKII) from the same tissue selec- tively phosphorylates the DNA-synthesizing a subunit and the primase-associated y subunit of the replicative complex.However, in vitro phosphorylated DNA polymerase a did not display altered catalytic activities. Furthermore, dephosphorylation of DNA polymerase a with calf intestine alkaline phosphatase (CIAP) did not change the catalytic properties either. Since there is a cell -cycle-dependent phosphorylation and dephosphorylation of the DNA-polymerase-a -DNAprimase complex, but no obvious alteration in basic enzymic properties, protein phosphorylation seems to affect more indirect properties of the enzyme, such as a complex formation with accessory proteins or nuclear structures. EXPERIMENTAL PROCEDURES Mu teriulsCalf thymus DNA-polymerase-a -primase was purified to near homogeneity as described [22]; the purific...

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“…In in vitro reactions, it phosphorylates a set of basic nonhistone proteins [4], and HMG proteins 14 and 17 [5,6], or activates RNA polymerases I and II by phosphorylation [7,8]. Casein kinase II, which is identical or closely related to protein kinase NII, modulates topoisomerase II activity [9].…”

Section: Introductionmentioning

confidence: 99%