Andrew Pigon

A majority of casein kinase II α subunit is tightly bound to intranuclear components but not to the β subunit

Nuclear casein kinase II (CK II) was purified from an epithelial cell line ofChironomus tentans and characterized. The intracellular distribution of CK II and its two intracellular subunits (α and β) was analysed by immunoblotting. The apparent molecular weights of the α and β subunits were estimated to be 36 and 28 kDa, respectively. Like other purified CK II preparations, CK II fromChironomus tentans is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and by the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual α and β subunit pools could be detected. More than 85% of the total immunostainable α subunit and essentially all immunoreactive individual β subunit and heterooligomeric enzyme molecules were localised to the nucleus. Unexpectedly, more than 80% of this nuclear α subunit was insoluble in 0.35 M NaCl, while all individual β subunit and heterooligomeric enzyme molecules were solubilized under the same conditions. Of the 0.35 M NaCl soluble kinase fractions, the active multisubunit form of CK II precipitated in 50% (NH4)2SO4 and could thus be separated from the free β subunit, which precipitated at 60% and 80% (NH4)2SO4. These results suggest that a major portion of the nuclear CK II α subunit does not form heterooligomeric structures with the β subunit, but binds tightly to nuclear components.

Sequences translated by Balbiani ring 75S RNA in vitro are present in giant secretory protein from Chironomus tentans.

The 75S RNA originating in the large Balbiani rings 1 and 2 (BR1 and 2) was isolated and used for in vitro translation in the mRNA dependent reticulocyte lysate. Conditions (K+-concentration, temperature, time etc), were optimized for obtaining translation products of maximal size. Polypeptide chains up to about 500,000 D were obtained but no complete translation products. Tryptic fingerprints were performed on the in vitro products as well as on the secretory protein components nos. I and II+III labelled with 35S-methionine. There was a large degree of correspondence between the fingerprint of the in vitro product and that of component I but less to that of component II+III. The results suggest that 75S RNA with an origin in the BR1 and BR2 codes for the giant secretory protein component I.

Immune and injury responses in Cecropia pupae—RNA isolation and comparison of protein synthesis in vivo and in vitro

Abstract If pupae of Hyalophora cecropia are injected with live bacteria they respond by producing about ten immune proteins ( Boman , 1980). At least four of these proteins are bactericidal. A sham injection with a sterile salt solution gave rise to a related injury response which may be nearly similar in total protein synthesis but with only a low bactericidal activity. This difference in response was correlated to the amounts of some small basic antibacterial proteins. Inhibition of transcription by actinomycin D at different times showed that 5 hr were required for the formation of the RNA needed for the synthesis of the antibacterial proteins. A method was developed for the isolation of total RNA from immunized and injured pupae. The key step in this RNA purification was lanthanum precipitation followed by extractions with ethanol-EDTA. The RNA fraction obtained after affinity chromatography on poly-U-Sepharose was a complex mixture of high mol. wt species with no differences between the RNA obtained from immunized and injured pupae. Using a cell-free system for studying protein synthesis, total RNA from both types of pupae produced rather similar dose responses of 13–15 proteins. Translation of total RNA from an untreated pupa gave a partially different protein pattern. The regulatory implications of these results are discussed.

Phosphorylation dependence of the initiation of productive transcription of Balbiani ring 2 genes in living cells.

Using polytene chromosomes of salivary gland cells of Chironomus tentans, phosphorylation state-sensitive antibodies and the transcription and protein kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphosphorylated (pol IIO) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with 32Pi within minutes of its addition, and nuclear pol IIO is gradually converted to IIA; this conversion parallels the reduction in transcription of protein-coding genes. DRB also alters the chromosomal distribution of IIO: there is a time-dependent clearance from chromosomes of phosphoCTD (PCTD) after addition of DRB, which coincides in time with the completion and release of preinitiated transcripts. Furthermore, the staining of smaller transcription units is abolished before that of larger ones. The staining pattern of chromosomes with anti-CTD antibodies is not detectably influenced by the DRB treatment, indicating that hypophosphorylated pol IIA is unaffected by the transcription inhibitor. Microinjection of synthetic heptapeptide repeats, anti-CTD and anti-PCTD antibodies into salivary gland nuclei hampered the transcription of BR2 genes, indicating the requirement for CTD and PCTD in transcription in living cells. The results demonstrate that in vivo the protein kinase effector DRB shows parallel effects on an early step in gene transcription and the process of pol II hyperphosphorylation. Our observations are consistent with the proposal that the initiation of productive RNA synthesis is CTD-phosphorylation dependent and also with the idea that the gradual dephosphorylation of transcribing pol IIO is coupled to the completion of nascent pol II gene transcripts.

The binding of the alpha subunit of protein kinase CK2 and RAP74 subunit of TFIIF to protein-coding genes in living cells is DRB sensitive.

In a previous report, we documented that a major portion of the nuclear protein kinase CK2α (CK2α) subunit does not form heterooligomeric structures with the β subunit, but it binds tightly to nuclear structures in an epithelial Chironomus cell line [1]. We report here that the CK2α, but not β, subunit is co-localized with productively transcribing RNA polymerase II (pol II) on polytene chromosomes of Chironomus salivary gland cells. Likewise, the RAP74 subunit of TFIIF, a potential substrate for CK2, is co-localized with pol II. The occupancies of chromosomes with the CK2α and RAP74 subunits are sensitive to DRB, an inhibitor of pol II-based transcription and the activity of CK2 and pol II carboxyl-terminal kinases. DRB alters the chromosomal distribution of the CK2α and RAP74 subunits: there is a time-dependent clearance from the chromosomes of CK2α and RAP74 subunits, which coincides in time the completion and release of preinitiated transcripts after addition of DRB. The results suggest that both the CK2α and RAP74 subunits travel with the elongating pol II molecules along the DNA template during the entire transcription cycle. No detectable re-association of CK2α and RAP74 with the promoters takes, however, place after the completion of the preinitiated transcripts in the presence of DRB. In contrast, the binding of hypophosporylated pol II and TFIIH to the active gene loci is not abolished by the DRB regimen. Our data are consistent with the possibility that in living Chironomus salivary gland cells, DRB interferes with the recruitment of TFIIF, but not of TFIIH, to the promoter by interference with the activity of the CK2α subunit enzyme and phosphorylation of RAP74 and thereby DRB blocks transcription initiation. (Mol Cell Biochem 191: 149–159, 1999)

Selective repression of RNA polymerase II by microinjected phosvitin.

We have used a microinjection technique to examine whether injected phosvitin, in its capacity as substrate for casein kinase NII, could compete out the endogeneous phosphorylation of some nuclear phosphoproteins with regatory potential and thereby interfere with the activity of RNA polymerase II. Phosphorylation, which utilizes ATP as phosphate donor, was separated from phosphorylation which uses GTP. Phosvitin introduced into nuclei of salivary gland cells becomes phosphorylated by the endogeneous nuclear protein kinase(s) and incorporates phosphates from ATP as well as from GTP. The phosphorylation of nuclear proteins and phosvitin is heparin-sensitive, indicating that they are phosphorylated by casein kinase NII. Microinjected phosvitin does not seem to affect the incorporation of phosphate groups from ATP into nuclear proteins, but protein phosphorylation by GTP is influenced. Apart from a minor overall reduction of 32P-incorporation, the phosphorylation of a 42 kDa nuclear protein, a putative transcription stimulatory factor, and of a 115 kDa nuclear protein was competed out by 70%–80% compared with the control value obtained in the absence of phosvitin. Parallel analyses of DNA transcription in phosvitin-injected nuclei showed that the RNA polymerase II-mediated synthesis of hnRNA and Balbiani ring RNA was diminished by 80% and 90%, respectively. In contrast, the transcription of nucleolar pre-ribosomal 38 S RNA by RNA polymerase I remained unaffected. The inhibitory effect of injected phosvitin could be reversed by in vitro phosphorylation of phosvitin prior to injection, using isolated nuclei as source of protein kinase(s). Taken together, the results suggest a causal relationship between the modification of the GTP-dependent phosphorylation of specific nonhistone proteins and the activity of RNA polymerase II.

Fractionation and characterization of rapidly phosphorylated nuclear proteins in salivary gland cells of Chironomus tentans

Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with 32Pi. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of 32P) and not sedimentable (supernatant) material. About 90% of 32P in the supernatant fraction was sedimentable at 100,000 g (“disperse chromatin”). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of 32P. The 32P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the 32P-labeled nuclear proteins into 12 major bands in the Mr range of 12,000–120,000. The incorporation of 32P into most bands reached a steady-state within 5–10 min of incubation with 32Pi and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an Mr 95,000 phosphoprotein. Two of the fastest moving 32P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving 32P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.

Mitochondrial distribution and ATP levels in Chironomus salivary gland cells as related to growth, metabolic activity, and atmospheric oxygen tension

During larval development of the dipteran Chironomus tentans its salivary gland cells increase in volume and become highly polytenized. A part of this growth process, from the early second instar to the late fourth instar, which represents a 130-fold increase in cell volume, was investigated here and found to be accompanied by changes in the intracellular distribution of mitochondria. In very young animals the mitochondria were scattered throughout the cytoplasm but with increasing cell size they became concentrated in a narrow basal zone between infoldings of the plasma membrane until at the end of development essentially all of them were localized in this zone. ATP-ADP-AMP concentrations did not change with increased cell size during these developmental phases. However, inhibition of total or, predominantly, secretory protein synthesis in late fourth larval instar animals in order to decrease energy consumption resulted in striking increases in ATPADP ratios. Concomitantly mitochondria became distributed in the whole endoplasm and the number of mitochondria in the basal zone decreased. On the other hand, when young animals with scattered mitochondria were exposed to low oxygen pressure, all the mitochondria moved into the basal zone. Our observations support the view that the increased accumulation of mitochondria in the basal zone during growth of the salivary gland cells represents an adaptation to optimize oxygen supply for ATP production. This arrangement is necessary when cell size becomes extreme or oxygen supply short, unless the consumption of ATP is decreased. The results show that mitochondria are able to shift their intracellular position in an apparently functionally meaningful way.

The rapidly phosphorylated chromosomal 42-kDa protein is a subunit of larger protein complexes

We have isolated, purified and characterized a 42-kDa phosphoprotein which has been found to be preferentially associated with active gene loci of salivary gland cells of Chironomus tentans. The rapidly phosphorylated form of this protein could be extracted with 0.2 M NaCl. Chromatographic analysis by gel filtration revealed that a significant fraction of labelled 42-kDa polypeptide elutes with an apparent molecular mass of 150 to 200 kDa. The result suggests that a portion of the phosphorylated 42-kDa polypeptide in native state forms a multisubunit protein complex consisting of rapidly phosphorylated 42-kDa polypeptide chains alone.

Analysis of the structural relationship between the DNA-binding phosphoproteins pp42, pp43 and pp44 by in situ peptide mapping

A structural homology is established between three DNA-binding phosphoproteins located in the 42 to 44 kDa range, referred to as pp42, pp43 and pp44, from Chironomus tentans salivary gland cells by in situ peptide mapping. The staining patterns of pp42, pp43 and pp44 which resulted from digestion with Stapholycoccus aureus V8, trypsin or papain proteases show the presence of 8 to 15 spots majority of which have identical mobility. In the patterns of the digests generated by treatments with trypsin about 10 spots appear in common between any pair of the protein substrates. In addition, each pattern includes two to three peptides of mobility not present in the other. Thus the peptide mapping of pp42, pp43 and pp44 based on the staining patterns of proteolytic digests suggest the existence of structural homology between the three unlabelled substrates. The proteolytic peptides carrying the rapidly turning over phosphate groups form markedly different electrophoretic patterns than the unlabelled peptides visualized by staining. Treatment of 32P-labelled pp42, pp43 and pp44 with V8 generates only one labelled fragment in the 30 kD range. The cleavage patterns of pp44 produced by chymotrypsin or papain contain seven to ten labelled fragments while those of pp42 and pp43 contain only two. The 32P-labelled tryptic peptides of pp42, pp43 and pp44 exhibit a ladder pattern for each substrate which probably arise by a consecutive removal of 25 to 35 amino acid residues from the primary digestion products pp29, pp29.5 and pp30 by cleavage of four to five putative interdomain regions. The possibility that these three structurally related phosphoproteins belong to the category of transcription factors is discussed.