The proline-rich homeodomain protein (PRH) plays multiple roles in the control of gene expression during embryonic development and in the adult. Vascular endothelial growth factor (VEGF) is a mitogen that stimulates cell proliferation and survival via cell surface receptors including VEGFR-1 and VEGFR-2. VEGF signaling is of critical importance in angiogenesis and hematopoiesis and is elevated in many tumors. Here we show that PRH binds directly to the promoter regions of the Vegf, Vegfr-1, and Vegfr-2 genes and that in each case PRH represses transcription. We demonstrate that overexpression or knockdown of PRH directly impinges on the survival of both leukemic and tumor cells and that the modulation of VEGF and VEGF receptor signaling by PRH mediates these effects. Our findings demonstrate that PRH is a key regulator of the VEGF signaling pathway and describe a mechanism whereby PRH plays an important role in tumorigenesis and leukemogenesis.

The proline-rich homeodomain protein (PRH/Hex) regulates transcription by binding to specific DNA sequences and regulates mRNA transport by binding to translation initiation factor eIF4E. Protein kinase CK2 plays multiple roles in the regulation of gene expression and cell proliferation. Here, we show that PRH interacts with the β subunit of CK2 in vitro and in cells and that CK2 phosphorylates PRH. Phosphorylation of PRH by CK2 inhibits the DNA binding activity of this protein and dephosphorylation restores DNA binding indicating that this modification acts as a reversible switch. We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177. Site-directed mutagenesis demonstrates that mutation of either of these residues to glutamic acid partially mimics phosphorylation but is insufficient to completely block DNA binding whereas an S163E/S177E double mutation severely inhibits DNA binding. Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells. Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner.

Protein kinase CK2 promotes cell survival and the activity of this kinase is elevated in several cancers including chronic myeloid leukaemia. We have shown previously that phosphorylation of the Proline-Rich Homeodomain protein (PRH/Hhex) by CK2 inhibits the DNA-binding activity of this transcription factor. Furthermore, PRH represses the transcription of multiple genes encoding components of the VEGF-signalling pathway and thereby influences cell survival. Here we show that the inhibitory effects of PRH on cell proliferation are abrogated by CK2 and that CK2 inhibits the binding of PRH at the Vegfr-1 promoter. Phosphorylation of PRH by CK2 also decreases the nuclear association of PRH and induces its cleavage by the proteasome. Moreover, cleavage of phosphorylated PRH produces a stable truncated cleavage product which we have termed PRHΔC (HhexΔC). PRHΔC acts as a transdominant negative regulator of full-length PRH by sequestering TLE proteins that function as PRH co-repressors. We show that this novel regulatory mechanism results in the alleviation of PRH-mediated repression of Vegfr-1. We suggest that the re-establishment of PRH function through inhibition of CK2 could be of value in treatment of myeloid leukaemias, as well as other tumour types in which PRH is inactivated by phosphorylation.

Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α′ catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix.Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected. Protocol I. Preparation for fractionationThis section describes the preparation of poly-L-Lysine coated microscope coverslips and the attachment of cells prior to fractionation. If required the cells can be transiently transfected with protein expression vectors either before or after attachment. 2. Coat clean coverslips with poly-L-Lysine by incubating them in the solution for at least 1 hour on a rocking platform at 22°C. 3. Wash the coated coverslips with sterile distilled water twice and follow with a single wash with 96% ethanol. 4. Air dry the coated coverslips on a piece of filter paper and keep them in a dry container for future use (once dry they can be stacked). A. Preparation of poly-L-1. Place a poly-L-Lysine coated coverslip in a well in a 6-well plate with the coated surface facing up. 2. Seed K562 cells at a density of 7x10 5 cells/well in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and PS (penicillin 100units/ml, streptomycin 100mg/ml). In the case of K562 cells transient transfection can be performed using electroporation (0.4cm electroporation cuvettes with 1x10 7 cells in 200μl of media at 250V / 975μF) before seeding the cells. 3. Incubate the cells for 24 hours at 37°C in 5% CO2. 4. Pour off the medium and wash the cells twice with ice cold phosphate buffered saline (PBS). 5. Follow with the subcellular fractionation protocol.1. Place an uncoated coverslip in a well in a 6-well plate. 2. Wash twice in PBS prewarmed at 37°C. 3. Detach the cells by digesting with trypsin (0.03% EDTA, 0.25% trypsin) ...

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein–DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents.

A runaway PRH/HHEX-Notch3 positive feedback loop drives cholangiocarcinoma and determines response to CDK4/6 inhibition.

Background: The incidence of cholangiocarcinoma (CCA) is increasing worldwide and current single chemotherapeutic drug treatments are ineffective. CX-4945 and cisplatin are currently in clinical trial for CCA treatment. Materials and Methods: We assessed the effects of the sequence of administration of CX-4945 and cisplatin applied in combination treatments on their efficacy in CCA cells in vitro. CCA cell viability was examined using 3-( 4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Apoptosis was examined using flow cytometry. The percentage of cells positive for phosphorylated H2A histone family member X (γ-H2AX) were measured using both flow cytometry and immunofluorescence. Results: CCA cell viability was reduced to 50% after 24 h of treatments with CX-4945 and cisplatin as single agents. Interestingly, treatment with cisplatin 6 h prior to CX-4945 treatment induced significantly more DNA damage and apoptosis than CX-4945 treatment followed by cisplatin. Unexpectedly, CX-4945 treatment followed by cisplatin was less effective than single treatment in RMCCA-1 CCA cells. In addition, a 1:1 ratio of each drug was the most effective combination in these cells. Conclusion: These data demonstrate that the combination of CX-4945 and cis platin acts additively when cisplatin is applied first, at least in part due to increased DNA damage and apoptosis. Furthermore, treatment with CX-4945 prior to cisplatin treatment reduces the efficacy of this drug combination in CCA cells.