casein-kinase-ii and Cell-Transformation--Viral

The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7's activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential.

Topics: Casein Kinase II; Cell Proliferation; Cell Transformation, Viral; DNA-Binding Proteins; Female; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Oncogene Proteins, Viral; Phenotype; Phosphorylation; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Autonomous parvoviruses induce severe morphological and physiological alterations in permissive host cells, eventually leading to cell lysis and release of progeny virions. Viral cytopathic effects (CPE) result from specific rearrangements and destruction of cytoskeletal micro- and intermediate filaments. We recently reported that inhibition of endogenous casein kinase II (CKII) protects target cells from parvovirus minute virus of mice (MVM)-induced CPE, pointing to this kinase as an effector of MVM toxicity. The present work shows that the parvoviral NS1 protein mediates CKII-dependent cytoskeletal alterations and cell death. NS1 can act as an adaptor molecule, linking the cellular protein kinase CKIIalpha to tropomyosin and thus modulating the substrate specificity of the kinase. This action results in an altered tropomyosin phosphorylation pattern both in vitro and in living cells. The capacity of NS1 to induce CPE was impaired by mutations abolishing binding with either CKIIalpha or tropomyosin. The cytotoxic adaptor function of NS1 was confirmed with fusion peptides, where the tropomyosin-binding domain of NS1 and CKIIalpha are physically linked. These adaptor peptides were able to mimic NS1 in its ability to induce death of transformed MVM-permissive cells.

Topics: Casein Kinase II; Cell Line; Cell Transformation, Viral; Cytopathogenic Effect, Viral; Enzyme Activation; Humans; Minute Virus of Mice; Phosphorylation; Protein Binding; Tropomyosin; Viral Nonstructural Proteins; Virus Internalization

In mammalian cells, the guanine nucleotide exchange factor (GEF, eIF-2B) plays a major role in the regulation of initiation of protein synthesis. It catalyzes the exchange of eukaryotic chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the recycling of eIF-2 during polypeptide chain initiation. We used the Friend virus-transformed murine erythroleukemia (MEL) cell system to elucidate the translational regulatory processes that occur during growth and hexamethylene bisacetamide (HMBA)-induced cell differentiation. GEF activity is increased during growth and decreased during MEL cell differentiation, and this parallels the overall changes in protein synthesis during this period. Inhibition of GEF activity in induced cells may occur indirectly by phosphorylation of the alpha-subunit of eIF-2. However, the decrease in GEF activity in induced cells cannot be reversed by increasing the concentration of eIF-2-GDP added as a substrate in the GEF assay. This is diagnostic for the presence of eIF-2 alpha(P)-GDP in cell lysates and suggests that regulation of GEF activity may occur by one or more mechanisms other than eIF-2(alpha) phosphorylation. We have previously shown that the activity of GEF may be influenced directly by phosphorylation with casein kinase II (CK-II) of the 82-kD subunit of the factor. CK-II activity parallels the changes in GEF activity and the rate of protein synthesis during growth and differentiation of MEL cells. Addition of 1mM spermidine, a stimulator of CK-II but not of purified GEF, in induced MEL cell extracts enhances both CK-II and GEF activities approximately 48 and 32%, respectively. The results presented suggest that the inhibition of protein synthesis during MEL cell differentiation may be linked to the decreased CK-II and GEF activities.

Topics: Acetamides; Animals; Antineoplastic Agents; Casein Kinase II; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Viral; Clone Cells; Eukaryotic Initiation Factor-2; Friend murine leukemia virus; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Leukemia, Erythroblastic, Acute; Methionine; Mice; Models, Biological; Neoplasm Proteins; Protein Biosynthesis; Protein Serine-Threonine Kinases; Sulfur Radioisotopes; Tumor Cells, Cultured

The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation of T antigen by the associated kinase is reduced whereas a p34cdc2-kinase-specific peptide has no influence. In addition, the T-antigen-associated protein kinase can use GTP and ATP as phosphate donors. These properties together with the observation that immunopurified T antigen can be phosphorylated by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed cells provide evidence for a close correlation between cell transformation and phosphorylation of T antigen by the associated protein kinase.

Topics: Amino Acid Sequence; Antigens, Polyomavirus Transforming; Casein Kinase II; Cell Line, Transformed; Cell Transformation, Viral; Humans; Molecular Sequence Data; Mutation; Peptides; Phosphorylation; Protein Serine-Threonine Kinases; Recombinant Proteins; Simian virus 40; Substrate Specificity; Temperature

Protein phosphorylation plays an important role in the regulation of cellular growth and proliferation and is thus thought to play a role in tumorigenesis. It has previously been reported that cells transformed by human cytomegalovirus (HCMV) contain two to four fold higher than normal levels of protein phosphorylation on serine and threonine residues, and two to six fold higher than normal levels of a casein kinase activity. We have now identified the major casein kinase activity found elevated in HCMV transformed cells as casein kinase type II; identification of this kinase was necessary in order to begin to define its role in HCMV mediated morphological transformation. Most of the differences in casein kinase II activity between normal and HCMV transformed cells were explained by differences in casein kinase II protein levels. This represents the first report concerning the elevation of casein kinase II activity in cells transformed by human cytomegalovirus.

Topics: Amino Acid Sequence; Casein Kinase II; Cell Line; Cell Transformation, Viral; Cytomegalovirus; Cytoplasm; Humans; Immunoblotting; Molecular Sequence Data; Peptides; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Substrate Specificity

The tumor suppressor protein p53 is phosphorylated at a C-terminal residue (serine 386 in mouse p53) by the protein kinase CK2. Phosphorylation by CK2 activates the specific DNA binding function of p53 and stimulates its ability to suppress cellular growth. Previous reports have suggested that phosphorylation of p53 at the CK2 site is stimulated in cells expressing the large tumor antigen (T antigen) of simian virus 40 (SV40). To test this idea, we have expressed a C-terminal p53 "mini-protein" which comprises amino acids 154-387 of mouse p53 and therefore lacks the heavily phosphorylated N-terminus. In addition, the serine 309 phosphorylation site (targeted by cyclin-dependent kinases) has been mutated to encode alanine. We have expressed the p53 mini-protein in mammalian cells and shown by phosphopeptide mapping that it is phosphorylated at a single physiological phosphorylation site, serine 386. Using this mini-protein as a cellular target for CK2, we have shown that phosphorylation of p53 by CK2 is not affected by the presence of T antigen. The p53 mini-protein is likely to be a useful tool with which to probe the regulation of p53 phosphorylation by CK2 in response to other factors which influence cell growth.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Antigens, Polyomavirus Transforming; Casein Kinase II; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; DNA; Fibroblasts; Mice; Molecular Sequence Data; Peptide Fragments; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Rats; Recombinant Fusion Proteins; Simian virus 40; Tumor Suppressor Protein p53