Simian Virus 40 Gene A Regulates the Association Between a Highly Phosphorylated Protein and Chromatin and Ribosomes in Simian Virus 40-Transformed Cells

The species of proteins associated with chromatin and ribosomes of simian virus 40 (SV40)-transformed and untransformed monkey, mouse, and rat cells have been compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after phosphorylation of these proteins either in vivo or in vitro. In vitro phosphorylation was carried out by protein kinase associated with these organelles and [gamma-(32) P]ATP as the phosphoryl donor. The reaction products contained both phosphoserine and phosphothreonine in approximately equal amounts. The electrophoretic analysis of the phosphorylated proteins revealed that the highly phosphorylated protein with a molecular weight of approximately 90,000 (90K protein) was associated with chromatin and ribosomes from transformed cells but not from untransformed cells. The 90K protein could be extracted from chromatin and ribosomes with 0.5 to 1.0 M NaCl or KCl. The 90K protein was still associated with the runoff ribosomes prepared by the puromycin reaction of the post-mitochondrial supernatant in the protein-synthesizing system. In vitro phosphorylation of chromatin and ribosomes from SV40 tsA-transformed mouse and rat cells indicated that the amounts of 90K protein associated with these organelles decreased greatly when the cells were cultivated at the restrictive temperature. A similar temperature-dependent decrease in the amount of (32)P-labeled 90K protein was observed in nonhistone chromosomal and ribosome-associated protein fractions prepared from SV40 tsA-transformed cells labeled with [(3)H]leucine and [(32)P]orthophosphate in vivo. In vitro phosphorylated 90K protein in nonhistone chromosomal and ribosome-associated proteins extracted with high salt was not immunoprecipitated with anti-SV40 T sera.     

Cell density-dependent increase in chromatin-associated ADP-ribosyltransferase activity in simian virus 40-transformed cells.

ADP-ribosyltransferase activity associated with chromatin is two- to tenfold higher in simian virus 40 (SV40)-transformed cells than in untransformed cells. When confluent transformed cells were subcultured, their specific enzyme activity first decreased two- to fourfold and the rapidly increased during the logarithmic phase of growth. This increase ceased or slowed down when the cells entered the stationary phase. In contrast, the activity in the untransformed cells remained low throughout the growth cycle. In SV40tsA-transformed cells (ts = temperature sensitive), this density-dependent increase in the enzyme activity was observed when the cells were cultivated at the permissive temperature, whereas the activity remained low at the restrictive temperature. The enzyme activity did not increase during induction of cellular DNA synthesis in quiescent cells either by addition of fresh medium or by infection with SV40. The chromatin-associated enzyme activity extracted with 1 m NaCl was eluted together with almost all the DNA-binding proteins from a phosphocellulose column with 0.6 m NaCl. The enzyme activity in this fraction from transformed cells, measured with or without added DNA and histones, was higher than that in a similar fraction from untransformed cells, reflecting the difference in the original activities present in the nuclei of these cells. The chain lengths of poly(ADP-ribose) formed by chromatin from SV40-transformed and untransformed cells were not significantly different. These results suggest that the number of initiation sites for ADP-ribosylation is increased in the chromatin of SV40-transformed cells compared to that of untransformed cells.     

Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon

The phosphorylation activity associated with a neurofilament-enriched cytoskeletal preparation isolated from the squid giant axon has been studied and compared to the phosphorylation activities in intact squid axoplasm. The high molecular weight (greater than 300 kDa) and 220-kDa neurofilament proteins are the major endogenous substrates for the kinases in the axoplasm and the neurofilament preparation, whereas 95- and less than 60-kDa proteins are the major phosphoproteins in the ganglion cell preparation. The squid axon neurofilament (SANF) protein kinase activity appeared to be both cAMP and Ca2+ independent and could phosphorylate both casein (Km = 40 microM) and histone (Km = 180 microM). The SANF protein kinase could utilize either ATP or GTP in the phosphotransferase reaction, with a Km for ATP of 58 microM and 129.4 microM for GTP when casein was used as the exogenous substrate; and 25 and 98.1 microM for ATP and GTP, respectively, when the endogenous neurofilament proteins were used as substrates. The SANF protein kinase activity was only slightly inhibited by 2,3-diphosphoglycerate and various polyamines at high concentrations and was poorly inhibited by heparin (34% inhibition at 100 micrograms/ml). The failures of heparin to significantly inhibit and the polyamines to stimulate the SANF protein kinase indicate that it is not a casein type II kinase. The relative efficacy of GTP as a phosphate donor indicates that SANF protein kinase differs from known casein type I kinases. Phosphorylated (32P-labeled) neurofilament proteins were only slightly dephosphorylated in the presence of axoplasm or stellate ganglion cell supernatants, and the neurofilament-enriched preparation did not dephosphorylate 32P-labeled neurofilament proteins. The axoplasm and neurofilament preparations had no detectable protein kinase inhibitor activity, but a strong inhibitor activity, which was not dialyzable but was heat inactivatable, was found in ganglion cells. This inhibitor activity may account for the low phosphorylation activity found in the stellate ganglion cells and may indicate inhibitory regulation of SANF protein kinase activity in the ganglion cell bodies.     

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases. (C) Proteins phosphorylated by ATP as well as GTP utilizing protein kinase(s) other than casein NII. Category B proteins can be subdivided into proteins phosphorylated in a heparin-resistant (B1) and heparin-sensitive (B2) manner. The phosphorylation of category C proteins may be heparin sensitive with ATP only (C1), heparin sensitive with GTP only (C2), heparin insensitive with both ATP and GTP (C3) or stimulated by heparin (C4).     

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.     

Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei.     

MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection.

Phosphopeptide analyses of the simian virus 40 (SV40) large tumor antigen (LT) in SV40-transformed rat cells, as well as in SV40 lytically infected monkey cells, showed that gel-purified LT that was not complexed to p53 (free LT) and p53-complexed LT differed substantially in their phosphorylation patterns. Most significantly, p53-complexed LT contained phosphopeptides not found in free LT. We show that these additional phosphopeptides were derived from MDM2, a cellular antagonist of p53, which coprecipitated with the p53-LT complexes, probably in a trimeric LT-p53-MDM2 complex. MDM2 also quantitatively bound the free p53 in SV40-transformed cells. Free LT, in contrast, was not found in complex with MDM2, indicating a specific targeting of the MDM2 protein by SV40. This specificity is underscored by significantly different phosphorylation patterns of the MDM2 proteins in normal and SV40-transformed cells. Furthermore, the MDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of MDM2 by SV40 is aimed at preventing MDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells.     

Regulation of amino acid and glucose transport activity expressed in isolated membranes from untransformed and SV 40-transformed mouse fibroblasts.

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and their SV 40-transformed derivatives were shown to catalyze carrier-mediated, intravesicular uptake of alpha-aminoisobutyric acid and D-glucose. Concentrative uptake of alpha-aminoisobutyric acid required the presence of a Na+-gradient (external greater than internal) and could occur independently of endogenous (Na+ + K+)ATPase activity. A K+ diffusion gradient (internal greater than external) in the presence of valinomycin, or the addition of the Na+ salt of a highly permeant anion, conditions expected to create an interior-negative membrane potential stimulated Na+-gradient-dependent uptake, suggesting this process is electrogenic. D-Glucose uptake was nonconcentrative and did not require ion gradients or metabolic conversion. Na+ gradient-dependent transport of alpha-aminoisobutyric acid was reduced both in initial rate and extent of uptake in vesicles from confluent untransformed cells and increased in those from SV 40-transformed cells, compared with activities observed in vesicles from proliferating untransformed cells. No changes in D-glucose carrier activity were observed when assayed at low glucose concentrations.     

Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.     

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

Summary Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.     

Activation of endogenous retroviral transcription in SV40-transformed mouse cells.

We have previously isolated a number of cDNA clones that correspond to mRNAs present at higher levels in SV40-transformed cells than in the untransformed parental cells (Scott, M.R.D., Westphal, K.-H. and Rigby, P.W.J. (1983) Cell 34, 557-567). We have now determined the nucleotide sequence of the archetypal Set 2 clone, pAG59, and can thus identify it as corresponding to the env gene of the endogenous, ecotropic C-type retrovirus of Balb/c mice, Emv-1. We have shown that in the subset of SV40-transformed cells that express the provirus both of the proteins encoded by env, gp70 and p15E, are synthesised and that the former is displayed on the cell surface. We discuss the significance of these observations for the biology of SV40 transformation.     

Adenosine 3',5'-monophosphate-dependent protein kinases of myocardial non-histone nuclear proteins.

Myocardial acidic non-histone nuclear proteins (NHPs) contain endogenous protein kinase activity. Phosphocellulose chromatography of purified NHPs identifies nine separate peaks of protein kinases which can phosphorylate both endogenous and exogenous substrates to a variable degree; endogenous NHPs are the best substrates. Cyclic AMP-stimulated protein kinase induced phosphorylation of endogenous and exogenous substrates; the extent of this stimulation varied according to the protein kinase fraction and substrate used. Cyclic AMP also enhanced NHP-induced stimulation of RNA polymerase activity. This enhancement was dependent on protein kinase-induced phosphorylation of NHPs since it was prevented by alkaline phosphatase pretreatment. It is concluded that nuclear protein kinases regulate myocardial RNA synthesis by enhancing phosphorylation of NHPs and that this regulation is under cyclic AMP control.     

Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53.

The cellular phosphoprotein p53 is presumably involved in simian virus 40 (SV40)-induced transformation. We have monitored changes in the state of phosphorylation of p53 from normal versus SV40-infected or -transformed cells. In normal cells, p 53 was hardly phosphorylated. Upon infection or transformation, a quantitative and qualitative increase in p53 phosphorylation was observed as revealed by two-dimensional phosphopeptide analysis. This increase was dependent on a functional large T antigen. In rat cells, enhanced phosphorylation of p53 resulted in conversion to a second, electrophoretically distinct form. In cells transformed with transformation-defective mutants, phosphorylation of p53 was reduced and conversion to form 2 was inefficient. These data suggest (i) that SV40 large T antigen induces or activates a protein kinase, one substrate of which is p53, (ii) that transformation-defective mutants are impaired in kinase induction, and (iii) that either a certain phosphorylation state of p53 or the SV40-induced kinase is critical for efficient transformation.     

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.     

Rous sarcoma virus-induced changes in the pattern of phosphorylation of non-histone nuclear proteins

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV).The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins.However, two-dimensional gel electrophoresis of the ³²P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2.The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60^src kinase.     

Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates

Summary The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examining competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s).The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP.Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.     

Death-associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis

Death-associated protein kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that is a drug discovery target for neurodegenerative disorders. Despite the potential profound physiological role of DAPK in neuronal function and pathophysiology, the endogenous substrate(s) of this kinase and the mechanisms via which DAPK elicits its biological action remain largely unknown. We report here that the mammalian 40S ribosomal protein S6 is a DAPK substrate. Results from immunoprecipitation experiments are consistent with endogenous DAPK being associated with endogenous S6 in rat brain. When S6 is a component of the 40S ribosomal subunit complex, DAPK selectively phosphorylates it at serine 235, one of the five sites in S6 that are phosphorylated by the S6 kinase family of proteins. The amino acid sequence flanking serine 235 matches the established pattern for DAPK peptide and protein substrates. Kinetic analyses using purified 40S subunits revealed a K(m) value of 9 microM, consistent with S6 being a potential physiological substrate of DAPK. This enzyme-substrate relationship has functional significance. DAPK suppresses translation in rabbit reticulocyte lysate, and treatment of neuroblastoma cells with a stimulator of DAPK reduces protein synthesis. In both cases, suppression of translation correlates with increased phosphorylation of S6 at serine 235. These results demonstrate that DAPK is a S6 kinase and provide evidence for a novel role of DAPK in the regulation of translation.     

Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.     

Replicon sizes in non-transformed and SV40-transformed cells, as estimated by a bromodeoxyuridine photolysis method

Replicon sizes were measured in Simian Virus 40 (SV40)-transformed and untransformed normal human, xeroderma pigmentosum (XP), and mouse 3T3 cells with an X-ray plus bromodeoxyuridine (BUdR) photolysis method. Replicon sizes in SV40-transformed cells were at least twice those in untransformed counterparts, but DNA fork displacement rates were only slightly increased.