Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

RNA metabolism in nuclei isolated from HeLa cells.

Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [gamma-32P]GTP and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four gamma-32P-labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation methylates the nucleolar RNA by utilizing S-adenosyl-L-methionine as a methyl-group donor.     

Formation of palmityl-[13'-32P]coenzyme A from [gamma-32P]ATP in mitochondrial extracts of guinea pig liver.

Investigations of the incorporation of ³²P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ-³²P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA¹ + palmityl-CoA ? palmityl-CoA¹ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′-³²P]CoA via the dephospho-CoA kinase reaction with exogenous [γ-³²P]ATP. The described preparation of palmityl-[3′-³²P]CoA and palmityl-[³⁵S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.     

Formation of deoxycytidine diphosphate-diglyceride by nuclei isolated from HeLa cells.

Isolated nuclei from HeLa cells synthesize dCDP-diglyceride from dCTP at the rapid rate of 5–10 nmol/20 min/10⁸ nuclei. The incorporation of dCTP into this phospholipid precursor is thus 10 to 20 times faster than the incorporation of dCTP into DNA, in vitro, under the same conditions. ATP, phosphatidic acid, and MgCl₂ are required for optimal synthesis of dCDP-diglyceride. The reaction is completely inhibited by the presence of 0.04% Triton N-101. Liponucleotide formation occurs equally well with dCTP or CTP in this system and competition studies suggest that a single enzyme catalyzes the formation of dCDP- and CDP-diglyceride.     

Decreased incorporation of the photoaffinity probe 8N3-[gamma-32P]-GTP into a 45kD protein in lung tumors

Photoincorporation of 8N3-[gamma-32P]-GTP into tissue and cell extracts was examined using gel electrophoresis and autoradiography. Decreased photoincorporation into a 45kD band was observed in extracts from mouse lung tumors as compared to normal mouse lung, and in extracts from lung tumor-derived cell lines when compared to isolated bronchiolar epithelial cells. Decreased 45kD photolabelling was also observed in extracts of S49 lymphoma cyc- cells (deficient in Gs alpha, a 45kD GTP binding protein of receptor-coupled adenylate cyclase) when compared to wild type S49 cells. This, and the observation that there was no cholera toxin-catalyzed ADP-ribosylation in the 45kD band of lung tumor extracts, suggests that the 45kD band contains Gs alpha.     

Evidence that Ser-82 is a unique phosphorylation site on vimentin for Ca2(+)-calmodulin-dependent protein kinase II.

We identified the sites on vimentin that are phosphorylated by Ca2(+)-calmodulin-dependent protein kinase II (CaM-kinase II). Sequential analysis of the purified phosphopeptides demonstrated that the sites are -Thr-Arg-Thr-Tyr-Ser(PO4)38-Leu-Gly-Ser-Ala- and -Val-Arg-Leu-Leu-Gln-Asp-Ser(PO4)82-Val-Asp-, which are located within the amino-terminal head domain of vimentin. For Ser-82 but not Ser-38, the proposed CaM-kinase II recognition amino acid sequence (Arg-X-X-Ser/Thr) was not found. Studies with a series of synthetic peptide analogs corresponding to Ser-82 and its surrounding amino acid sequence indicate that Asp-84 acts as an essential substrate specificity determinant for the Ser-82 phosphorylation by CaM-kinase II. The CaM-kinase II recognition site may be more extensive than heretofore determined.     

Concanavalin A- and calcium-dependent phosphorylation of a protein of 80 kDa in mouse lymphocytes rendered permeable to exogenously added [gamma-32P]ATP

The phosphorylation of a protein of 80 kDa in permeable mouse lymphocytes is shown to be dependent both on exogenously added calcium and on concanavalin A. Lymphocyte plasma membranes are rendered permeable to exogenously added [gamma-32P]ATP and other small molecules by treatment with 20 micrograms/ml alpha-lysophosphatidylcholine for 1 min on ice. Treated cells are permeable to Trypan blue dye and exhibit phosphatidylinositol turnover in response to concanavalin A stimulation. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, maximal phosphorylation of this protein occurs 5 min after addition of 20 microM calcium and 4 micrograms/ml concanavalin A. Exogenously added cyclic nucleotide cofactors do not enhance the phosphorylation of this 80 kDa protein, nor do inhibitors of calcium or calmodulin-dependent kinases suppress it, although in each case, other proteins are affected. In contrast, an inhibitor of the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), H-7, strongly suppresses the phosphorylation of the 80 kDa protein. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a known activator of protein kinase C, significantly increases the phosphorylation of the 80 kDa protein. Finally, this protein is phosphorylated at a serine residue. These results taken together suggest that it is a substrate for protein kinase C. The possibility that it may also be an element of the concanavalin A signal transduction mechanism is discussed.     

5-Methyltryptamine decreases net accumulation of 32P into the polyphosphoinositides from [gamma-32P]ATP in a cell-free system from blowfly salivary glands. Activation of breakdown of the newly synthesized [32P]polyphosphoinositides

Incubation of blowfly salivary gland homogenates with 30 microM [gamma-32P]ATP resulted in a rapid, Mg2+-dependent, synthesis of [32P]polyphosphoinositides and [32P]phosphatidic acid. 5-Methyltryptamine, in the presence of 10 microM guanosine 5'-(3-O-thio)trisphosphate, reduced the net accumulation of 32P label into phosphatidylinositol-4,5-P2 and phosphatidylinositol-4-P by 35 and 20%, respectively. 5-Methyltryptamine did not affect synthesis of [32P]phosphatidic acid. Phosphorylation of polyphosphoinositides was not affected by 5-methyltryptamine. In membranes labeled in vitro with [gamma-32P]ATP, 5-methyltryptamine stimulated a rapid breakdown of the [32P]polyphosphoinositides. These results indicate that in blowfly salivary gland homogenates, hormone stimulates breakdown of the newly synthesized polyphosphoinositides. In the presence of hormone, the rate of polyphosphoinositide synthesis does not compensate for the rate of polyphosphoinositide degradation.     

Casein kinase II and the tumor suppressor protein P53 associate in a molecular complex that is negatively regulated upon P53 phosphorylation.

Selective immunoisolation of P53 from Sf9 cells coexpressing wild-type P53 and casein kinase II yielded a preparation containing casein kinase II, thus suggesting that the two proteins may associate in a molecular complex in the intact cell. Such a complex could indeed be demonstrated in vitro between purified recombinant P53 and oligomeric casein kinase II and was shown to dissociate when P53 became phosphorylated by the kinase. This suggested that the P53 C-terminal domain, which contains the casein kinase II phosphorylation site was involved in the protein-protein interaction; this was confirmed by the fact that an anti-P53 monoclonal antibody directed to that domain inhibited the P53-casein kinase II association. Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit could phosphorylate P53, the formation of a stable P53-casein kinase II association required the presence of the beta subunit of the kinase. This was confirmed by immunoisolation of a P53-beta subunit complex from cells expressing both polypeptides. Although the biological significance of a reversible P53-casein kinase II molecular complex in the control of cell proliferation processes remains to be defined, these observations suggest the possibility of a novel mechanism regulating P53 and casein kinase II activities in the intact cell.     

Protein kinase C [micro] is regulated by the multifunctional chaperon protein p32

We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.     

Transcription of specific genes in isolated nuclei from HeLa cells in vitro.

Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of alpha-amanitin (1 mug/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45-S ribosomal precursor was completely resistant even to high concentrations of alpha-amanitin (150 mug/ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45-S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4-8 S) was synthesized by HeLa cell nuclei in the presence of 1 mug/ml alpha-amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 mug/ml) but clearly sensitive to high (150 mug/ml) concentrations of alpha-amanitin. Consequently the synthesis of all these small-molecular-weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5' end of the RNA molecule with the gamma-phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small-molecular-weight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45-S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.     

Histone F1 from rat thymus. Subfractionation and incorporation of [32P]phosphate in vitro

Four subfractions of histone F1 from rat thymus were obtained by preparative polyacrylamide-gel electrophoresis. These subfractions are closely related in primary structure but show marked differences in radioactivity when derived from rat thymus nuclei labelled in vitro with [(32)P]phosphate.     

Evidence that glucagon-mediated inhibition of acetyl-CoA carboxylase in isolated adipocytes involves increased phosphorylation of the enzyme by cyclic AMP-dependent protein kinase.

The kinetic parameters and phosphorylation state of acetyl-CoA carboxylase were analysed after purification of the enzyme by avidin--Sepharose chromatography from extracts of isolated adipocytes treated with glucagon or adrenaline. The results provide evidence that the mechanism of inhibition of acetyl-CoA carboxylase in adipocytes treated with glucagon [Zammit & Corstorphine (1982) Biochem. J. 208, 783-788] involves increased phosphorylation of the enzyme. Hormone treatment had effects on the kinetic parameters of the enzyme similar to those of phosphorylation of the enzyme in vitro by cyclic AMP-dependent protein kinase. Glucagon treatment of adipocytes led to increased phosphorylation of acetyl-CoA carboxylase in the same chymotryptic peptide as that containing the major site phosphorylated on the enzyme by purified cyclic AMP-dependent protein kinase in vitro [Munday & Hardie (1984) Eur. J. Biochem. 141, 617-627]. The dose--response curves for inhibition of enzyme activity and increased phosphorylation of the enzyme were very similar, with half-maximal effects occurring at concentrations of glucagon (0.5-1 nM) which are close to the physiological range. In general, the patterns of increased 32P-labelling of chymotryptic peptides induced by glucagon or adrenaline were similar, although there were quantitative differences between the effects of the two hormones on individual peptides. The results are discussed in terms of the possible roles of cyclic AMP-dependent and -independent protein kinases in the regulation of acetyl-CoA carboxylase activity and of lipogenesis in white adipose tissue.     

Zinc-induced tyrosine phosphorylation of hippocampal p60c-src is catalyzed by another protein tyrosine kinase.

Tyrosine phosphorylation of p60c-src induced by Zn2+ in rat hippocampal membranes is shown to inhibit Src tyrosine kinase activity. Zn2+ catalyzes the phosphorylation of p60c-src in the membranes but does not activate autophosphorylation of p60c-src immunoprecipitated with anti-Src monoclonal antibody. Moreover, the immunoprecipitated Src kinase has no Zn(2+)-induced activity in phosphorylation of exogenous substrate, enolase. Cyanogen bromide cleavage of p60c-src phosphorylated in the presence of Zn2+ yields a 4-kDa phosphopeptide corresponding to phosphorylation of a carboxy-terminal tyrosine residue of Src kinase. In conclusion, hippocampal membranes contain a Zn(2+)-stimulated protein tyrosine kinase capable of regulating the p60c-src activity.