CK2 (formerly termed "casein kinase 2") is a ubiquitous, highly pleiotropic and constitutively active Ser/Thr protein kinase whose implication in neoplasia, cell survival, and virus infection is supported by an increasing number of arguments. Here an updated inventory of 307 CK2 protein substrates is presented. More than one-third of these are implicated in gene expression and protein synthesis as being either transcriptional factors (60) or effectors of DNA/RNA structure (50) or translational elements. Also numerous are signaling proteins and proteins of viral origin or essential to virus life cycle. In comparison, only a minority of CK2 targets (a dozen or so) are classical metabolic enzymes. An analysis of 308 sites phosphorylated by CK2 highlights the paramount relevance of negatively charged side chains that are (by far) predominant over any other residues at positions n+3 (the most crucial one), n+1, and n+2. Based on this signature, it is predictable that proteins phosphorylated by CK2 are much more numerous than those identified to date, and it is possible that CK2 alone contributes to the generation of the eukaryotic phosphoproteome more so than any other individual protein kinase. The possibility that CK2 phosphosites play some global role, e.g., by destabilizing alpha helices, counteracting caspase cleavage, and generating adhesive motifs, will be discussed.

One-thousand-and-one substrates of protein kinase CK2?

https://escholarship.org/content/qt4431p9mq/qt4431p9mq.pdf?t=qeh5tn

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

/pdf/dynamic-sorting-of-nuclear-components-into-distinct-r9xsu3ob7e.pdf

Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

The transcriptional program of a human B cell line in response to Myc

Protein kinase CK2 (also termed casein kinase-2 or -II) is a ubiquitous Ser/Thr-specific protein kinase required for viability and for cell cycle progression. CK2 is especially elevated in proliferating tissues, either normal or transformed, and the expression of its catalytic subunit in transgenic mice is causative of lymphomas. CK2 is highly pleiotropic: more than 160 proteins phosphorylated by it at sites specified by multiple acidic residues are known. Despite its heterotetrameric structure generally composed by two catalytic (α and/or α’) and two non catalytic β-subunits, the regulation of CK2 is still enigmatic. A number of functional features of the β-subunit which could cooperate to the modulation of CK2 targeting/activity will be discussed.

Protein kinase CK2 ("casein kinase-2") and its implication in cell division and proliferation.

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity. The nucleolus in eukaryotic cells carries out most of the important reactions in ribosome biogenesis, including rDNA (rRNA genes) transcription, rRNA processing, and preribosome assembly (34, 47, 49, 50). Elements of nucleolar architecture mediate a tight temporal and topological orchestration of these processes. In higher eukaryotic cells, nucleoli exhibit a common organization consisting of three ultrastructurally distinct regions, the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The main body of the nucleolus is made up of the GC, embedded in this granular mass are several islets of rounded structures, FCs, each surrounded by a compact layer of the DFC. Tandemly repeated rRNA genes are clustered mostly in the FCs (42, 57), with transcription occurring largely at the boundary between the FC and the DFC (13, 20, 42, 58). Nascent rRNA transcripts are processed in the DFC (25, 38, 59). Some processing steps may also occur in the GC, together with the assembly of the mature rRNA and ribosomal proteins into preribosomal subunits (42, 47). In addition to rDNA, rRNA, and ribosomal proteins, the nucleolus harbors a large number of nonribosomal proteins and small nucleolar RNAs, which mediate the transcriptional and posttranscriptional reactions of ribosome biogenesis (49, 54). Many nucleolar proteins locate in particular nucleolar regions, which may reflect functional compart

https://journals.asm.org/doi/pdf/10.1128/MCB.19.12.8536

Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization.

We identified a novel human nucleolar phosphoprotein p130 (130 kDa) using a strategy for selecting monoclonal antibodies against nuclear proteins which oscillate in the cell cycle. p130 is localized in interphase nucleoli in a dotted manner. Complete extraction of p130 required a high concentration of salt (0.5 M NaCl) indicating that it binds firmly to the nucleolar components via ionic interaction. p130 is heavily phosphorylated, since alkaline phosphatase treatment converted the purified p130 into a 95 kDa product; this was further supported by the in vitro demonstration that cellular phosphatase and casein kinase II activities were responsible for the interchange of these two forms. Extracts of mitotic cells had lower concentrations of p130 compared to those of interphase cells suggesting that a proportion of p130 might be degraded during mitosis. Moreover, all the remaining p130 in mitotic cells was further phosphorylated, likely by a cdc2 kinase, resulting in increase in its solubility, and its dispersion throughout the entire cytoplasm. Thus, p130 in metaphase and anaphase cells was unable to be detected by immunofluorescence microscopy. At telophase, p130 reappeared and aggregated into a granular structure, resembling the prenucleolar bodies. These granules migrated from the nucleoplasm to the nucleoli in early G1-phase. Actinomycin D was able to induce segregation of p130-containing granules into the nucleoplasm, similar to the well-known behavior of the fibrillarin-containing granules, indicating that p130 is localized in the dense fibrillar component, a subnucleolar region for pre-rRNA synthesis and processing. The cDNA sequence of p130 revealed a remarkable feature, that a serine-rich stretch interspersed with acidic residues is repeated ten times. Such a characteristic is shared with a rat nucleolar phosphoprotein Nopp140, which is thought to shuttle between the nucleolus and the cytoplasm. Although p130 shows 74% identity to Nopp140, our observations suggest that during mitosis the functions of p130 are related to nucleologenesis.

Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis.

The Nucleolar Phosphoprotein P130 Is a GTPase/ATPase with Intrinsic Property to Form Large Complexes Triggered by F−and Mg2+


 Background: Interleukin-10 (IL-10) has demonstrated utility in aiding CD8 T cell-mediated tumor control in mouse models but phase 2 clinical trials with pegylated IL-10 have so far not been entirely successful. More recent studies revealed that another half-life-extended form of IL-10 (IL10-Fc) can rejuvenate terminally-exhausted CD8+ T cells to boost anti-tumor activity. We investigated whether combination of a PDL-1-blocking antibody with a C-terminal IL-10 fusion may enhance anti-tumor immunity, and possibly achieve synergistic effects.
 Methods: Recombinant proteins were produced using an expiCHO system (Thermo Fisher). PDL-1 blocking activity was evaluated by ELISA, PD-1/PDL-1 signaling (Eurofins) and mixed lymphocyte reaction assays. IL-10 activity was evaluated by MC/9 cell proliferation and CD8 T cell activation and effector assays. Mouse CT26 colon cancer and EMT6 breast cancer studies employed the services of Oneness Biotech and Crown Bioscience, respectively.
 Results: To develop a bifunctional anti-PDL-1 antibody/IL-10 fusion protein, anti-human PDL-1 antibodies were first developed by screening an in-house human variable domain phage display library. Three different antibodies were then C-terminally-fused with human IL-10 polypeptide. All three showed high binding affinity to PDL-1 and blocked PD-1 signaling through PD-1/PDL-1 interaction in a dose-dependent manner. In CD4 T cell/dendritic cell co-cultures, the lead anti-PDL1/IL10 protein enhanced IL-2 and IFNγ production, and it enhanced IFNγ and granzyme B production from activated CD8 T cells as well as proliferation of MC/9 cells in an IL-10-dependent manner. Also, a mouse ‘surrogate’ version (BF11, using avelumab anti-PDL-1) showed superior efficacy compared to either IL10-Fc or anti-PDL1/TGFbR in a mouse CT26 colon tumor model, and correlated with increased serum levels of CXCL9 and IL-18. BF11 also allowed greater EMT6 tumor control than anti-PDL-1 alone or anti-PDL1/TGFbR fusion protein. Such BF11-treated mice also showed long-lasting immunity against a secondary EMT6 tumor challenge. This ability of BF11 to enhance tumor control was in part dependent on increased activity of tumor-resident cells as co-treatment with FTY720, which inhibits lymphocyte egress from secondary lymphoid organs, did not appear to reduce anti-tumor activity of BF11 in some, albeit not all, EMT6-tumor-bearing mice.
 Conclusions: Anti-PDL1/IL10 fusion protein not only targets PDL-1 for checkpoint blockade but also activates the IL-10 pathway to boost T cell response, presumably including revitalization of exhausted T cells as recently reported. Effective anti-tumor activities of a mouse surrogate fusion protein, BF11, are demonstrated in CT26 and EMT6 tumor models. Taken together, our findings support this dual mechanism-strategy to potentiate anti-tumor immunity.
 Citation Format: Huey-Wen Hsiao, Chih-Lun Hsiao, Pandelakis Koni, Hung-Kai Chen. A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4169.

Hung-Kai Chen

Papers

Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization.

Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis.

The Nucleolar Phosphoprotein P130 Is a GTPase/ATPase with Intrinsic Property to Form Large Complexes Triggered by F−and Mg2+

Abstract 4169: A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer immunotherapy