Effect of thyroid hormone on the phosphorylation of 110k proteins and the activity of protein kinase NII of rat liver nucleoli

When rat liver nucleoli were incubated with [gamma-32P] ATP, it was found that 110k proteins were predominantly phosphorylated. The phosphorylation of 110k proteins was decreased by thyroidectomy to about 80% of the normal level and restored 12 h after the injection of triiodothyronine. The activity of nucleolar protein kinase NII which is responsible for phosphorylation of 110k proteins was also increased by about 20-50% after injecting triiodothyronine into thyroidectomized rats. The hormone-stimulated enhancement in the phosphorylation of 110k proteins and protein kinase NII in nucleoli were abolished within 1 h after an injection of cycloheximide, without any change in the amount of the proteins.     

Phosphorylation of nucleolin by a nucleolar type NII protein kinase

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.     

Nucleolin, defective for MPF phosphorylation, localizes normally during mitosis and nucleologenesis

To determine what effect maturation promoting factor (MPF, p34 ^cdc2 kinase/cyclin B) phosphorylation has on nucleolin’s distribution during mitotic nucleolar disassembly and reassembly, we altered Chinese hamster ovary (CHO) nucleolin (the N protein) such that it cannot be phosphorylated by p34 ^cdc2 . As expected, the transiently expressed epitope-tagged N protein showed no apparent defect in nucleolar localization in interphase CHO cells, even after hypotonic shock and recovery to quickly disassemble and then reassemble interphase nucleoli. In mitotic CHO cells, the N protein localized to the perichromosomal sheath and the cytoplasm, as is typical for nucleolin. Similar to epitope-tagged wild-type nucleolin, the N protein also maintained its association with persistent nucleoli characteristic of mitotic Chinese hamster lung (Dede) cells. In synchronized HeLa cells, the N protein again localized to the perichromosomal sheath and the cytoplasm as nucleoli disassembled during prophase. In HeLa cell telophase, the N protein localized normally to nucleolus-derived foci within the cytoplasm and prenucleolar bodies within reforming nuclei. The observations indicate that MPF phosphorylation is not essential for nucleolin’s localizations to the perichromosomal sheath and the cytoplasm during prophase and metaphase, and that functional MPF phosphorylation sites are not essential for nucleolin’s localizations during nucleologenesis. Accepted: 15 April 1999     

Effect of dexamethasone on nucleolar casein kinase II activity and phosphorylation of nucleolin in lymphosarcoma P1798 cells.

Ribosomal RNA (rRNA) synthesis in murine P1798 lymphosarcoma cells is reversibly inhibited by glucocorticoids. The effects of dexamethasone upon nucleolin phosphorylation and upon the amount and activity of casein kinase II have been examined. P1798 cells were exposed to 0.1 microM dexamethasone for 36 h. Cells were labeled in vivo with [32P]orthophosphate followed by immunoprecipitation with anti-nucleolin antibody. Nucleolin phosphorylation was reduced by 60% in dexamethasone-treated cells. Nucleoli were isolated and labeled with [gamma-32P]ATP in vitro. Nucleolin protein was reduced to 40% of control in nuclei from dexamethasone-treated cells. Nucleolin phosphorylation was reduced to 20% of control. Nucleolar casein kinase II activity and protein were also reduced (30-55% and 35-50% of control, respectively) by treatment with dexamethasone. Cycloheximide (10 micrograms/ml for 3 h) reduced the amount and activity of casein kinase II, but did not cause a decrease in nucleolin protein. These observations are discussed relative to the hypothesis that glucocorticoids regulate the amount or activity of proteins of short biological half-life that are involved in the regulation of rRNA synthesis.     

Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.     

The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.     

RNA SYNTHESIS IN CULTURED CHICK EMBRYO CELLS IN GROWING AND CONFLUENT PHASES

RNA synthesis at the growing phase in monolayer cultures of chick embryo fibroblasts was compared with that at confluent phases by zonal sedimentation, base composition and hybridization experiments. The nuclei were isolated by treatment with Nonidet p-40. The ratio of RNA/DNA in isolated nuclei was higher at the growing phase than that of confluent. The rate of RNA synthesis was reduced in the cells at confluent phase to 15.1% of that at the growing phase. The sucrose density gradient sedimentation pattern of nuclear RNA was on the whole the same in both phases. According to the distribution of ¹⁴C-uridine incorporated into nuclear RNA, 45S ribosomal precursor RNA was more distinct for the growing cell, while the radioactivities were found to be polydispersed, including the RNA which sedimented faster than 28S RNA in the cells at confluent phase. The base compositions and hybridization analyses indicated that ribosomal RNA was synthesized more actively in the growing cells. About 50% of newly synthesized RNA was ribosomal in the growing cells but 35% in the confluent.
It was found that newly synthesized 18S and 28S ribosomal RNAs appeared in cytoplasm after 21 and 33 min lag periods respectively. These times were exactly same in both growing and confluent phases.     

Spermine Stimulation of a Nuclear NII Kinase from Pea Plumules and Its Role in the Phosphorylation of a Nuclear Polypeptide

We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684) In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg²⁺ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [γ-³²P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.     

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.     

Isolation and localization of phosphoprotein pp 135 in the nucleoli of various cell lines

Phosphoprotein pp 135 is one of the dominant proteins endogenously phosphorylated in cellular sonicates during short-time exposure to [gamma-32P]ATP. Mouse cells growing exponentially show the highest pp 135 level as determined by endogenous phosphorylation and immunobinding assays. Disruption of cells in the absence of calcium at low magnesium concentration renders more than 90% pp 135 into the cytosolic fraction. A five-step purification yields greater than 95% pure pp 135. The cellular location of pp 135 was determined with a rat anti-(mouse pp 135) serum by immunofluorescence in mouse cell lines and cryostat sections of normal mouse tissue. We observed fluorescence predominantly of nucleolar structures, confirmed by studies of isolated nuclei and nucleoli. Cross-reacting nucleolar phosphoproteins were identified in cell lines of other species with molecular masses of 128 kDa (human), 135 kDa (hamster) and 118 kDa (Drosophila). Endogenous phosphorylation of pp 135 investigated with purified mouse nucleoli showed optimal activity at isotonicity, pH 7.3, in the presence of 10 mM magnesium ions.