Protein kinase CK2 inactivates PRH/Hhex using multiple mechanisms to de-repress VEGF-signalling genes and promote cell survival

Abstract

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.

Figure 1.

CK2 alleviates the repression of VSP genes by PRH. (A) Vegfr-1 mRNA levels in K562 cells 48 h post-transfection with empty vector (1), plasmid expressing Myc-PRH (5 µg) (2) or plasmids expressing Myc-PRH (5 µg) and HA-CK2 subunits (3 µg each) (3–5). mRNA levels were determined by qPCR and compared to Gapdh. Mean and standard deviation (M + SD), n = 5. ** indicates P < 0.005, ns indicates not significant. (B) Vegf mRNA levels in K562 cells transfected as in (A) determined as above. M + SD, n = 5. (C) PRH protein levels after transfection with scrambled vector control (SVC)(1) or PRH shRNA (2) followed by selection using puromycin. Endogenous PRH was detected using PRH-specific antibodies. (D) Vegfr-1 mRNA levels after PRH KD. Control and PRH KD cells were treated with 80 µM DMAT for 24 h prior to mRNA isolation. White bars represent SVC shRNA targeted control cells and grey bars PRH shRNA targeted cells. M + SD, n = 3. **P < 0.005, ns—not significant. (E) Vegf mRNA levels in the cells from (D).

Figure 2.

CK2 alleviates the inhibition of cell proliferation by PRH. (A) K562 cells were transfected with plasmids expressing Myc-PRH (5 µg) or Myc-PRH (5 µg) and CK2 subunits (3 µg each). Seventy-two hours post-transfection cells were counted using trypan blue exclusion. M + SD, n = 5. * indicates P < 0.02, ns—not significant. (B) K562 cells were transfected with 1 µg pEGFP alone (control), 1 µg pEGFP and 5 µg pMUG1-Myc-PRH (PRH) or 1 µg pEGFP, 5 µg pMUG1-Myc-PRH and 3 µg of plasmids expressing CK2α and CK2β. The cells were dual stained with propidium iodide (P.I.)/Annexin V (AV) (APC antibody) 24 h post-transfection and analysed by flow cytometry. Cells stained with both dyes are in late apoptosis. Cells stained for annexin V alone are in early apoptosis. The dot plot shows the percentage of live cells (PI−/AV−), necrotic cells (PI+), early apoptotic cells (AV+) and late apoptotic cells (AV+/PI+) after gating for GFP+ cells. Representative data. (C) The experiment shown in (C) was repeated three times and the graph shows the percentage of early and late apoptotic cells. * indicates P < 0.02, ns—not significant.

Figure 3.

CK2 abolishes DNA binding and the repression of VSP genes by PRH. (A) Vegfr-1 mRNA levels in K562 cells 48 h post-transfection with an EVC or plasmids expressing PRH, PRH CC and PRH EE either alone, or in conjunction with plasmids expressing CK2 subunits. mRNA levels were determined as in Figure 1. M + SD, n = 3. *P < 0.05, ns—not significant. (B) Western blot of whole-cell extracts from K562 cells transfected as in (A). (C) K562 cells were transfected as in (A) and chromatin was assessed for distribution of fragment size by electrophoresis on a 1% agarose gel. M = 1 kb Marker, chromatin from 2.5 × 10⁶ cells sonicated for 5 min (1) or 10 min (2), chromatin from 5 × 10⁶ cells sonicated for 5 min (3) or 10 min (4). Sonication of chromatin from 5 × 10⁶ cells results in fragments averaging <400 bp and these conditions were used in ChIP. (D) Upper panel—a cartoon of the Vegfr-1 genomic region, showing relative positions of the Vegfr-1 promoter (bent arrow), clusters of PRH-binding sites (filled boxes), and Vegfr-1 primer sequences used for ChIP. Lower panels- enrichment of Myc-PRH proteins bound Vegfr-1 primer sequences relative to input. Template DNA was precipitated using the Myc 9E10 antibody or IgG. M + SD, n = 4.

Figure 4.

CK2 reverses the inhibition of cell proliferation by PRH but not PRH CC. (A) K562 cells were transfected as in Figure 3A and 72 h post-transfection cells were counted as in Figure 1. M + SD, n = 5. **P < 0.005, ns—not significant. (B) K562 cells were co-transfected with a plasmid expressing GFP and an empty vector (EVC) or plasmids expressing GFP, PRH proteins and CK2 subunits. Twenty-four hours post-transfection the cells were dual stained as in Figure 2. The graph shows the fold change in the number of apoptotic cells (AV+/PI+) after gating for GFP+ cells. M + SD, n = 3. *P < 0.05, ns—not significant.

Figure 5.

A PRH phosphomimic shows altered intracellular localization. (A) K562 cells were transfected with vectors expressing Myc-tagged PRH, PRH CC or PRH EE and then fractionated into cytoplasmic and loosely held nuclear proteins (PN) and tightly held nuclear proteins (N). The extracts were western blotted for PRH using the Myc antibody (top panel). The blot was stripped and reprobed for Tubulin and Lamin A/C as controls for fractionation and loading. (B) K562 cells were transiently transfected as above and then adhered to polylysine coated coverslips. Top three rows, whole-cell images. Bottom three rows, cells treated with CSK buffer containing 0.1% SDS to remove cytoplasmic and loosely held nuclear proteins. DNA was stained with DAPI. Tubulin was visualized using an anti-Tubulin antibody and FITC-labelled secondary. PRH was visualized using the Myc 9E10 antibody and a TRITC-labelled secondary. Viewed using a Leica DM IRBE confocal microscope. (C) K562 cells were transfected with plasmids expressing Myc-PRH alone or Myc-PRH and HA-CK2 α and β subunits (as in Figure 1A). The cells were then fractionated into whole-cell extract (WC), PN and N fractions as in (part A above). The extracts were western blotted for PRH using the Myc antibody (top panel). Tubulin and Lamin A/C were used as controls for fractionation and loading.

Figure 6.

pPRH is rapidly cleaved by the proteasome to produce a stable product. (A) Untransfected K562 cells were treated with Anisomycin for the times indicated and then fractionated as in Figure 5. The extracts were western blotted for endogenous PRH using antibodies that recognize hypo-PRH or pPRH. The blot was stripped and reprobed for Lamin A/C and Tubulin as controls for fractionation and loading. (B) The experiment described in (A) was repeated using K562 cells treated with 40 µM anisomycin and the proteasome inhibitor MG132 (10 µM). (C) K562 cells were transiently transfected with plasmids expressing Myc-PRH (5 µg) or Myc-PRH (5 µg) and HA-CK2 subunits (3 µg each). Twenty-four hours post-transfection whole-cell extracts were western blotted for Myc-tagged proteins using the Myc9E10 antibody. The blot was stripped and reprobed for Tubulin as a control for loading. (D) K562 cells were transiently transfected with EVC or plasmids expressing Myc-PRH (5 µg), Myc-PRH EE (5 µg) and Myc-PRH CC (5 µg). Twenty-four hours post-transfection whole-cell extracts were western blotted for Myc-tagged proteins and Tubulin as a control for loading. (E) The ratio of PRHΔC to full-length PRH was determine from three independent experiments performed as in (D). *P < 0.05, (F) Vegfr-1 mRNA levels in K562 cells 48 h post-transfection with plasmids expressing Myc-PRH (5 µg) alone, Myc-PRHΔC EE (5 µg) alone or Myc-PRH (5 µg) and Myc-PRHΔC EE (1, 3 and 5 µg). mRNA levels were determined by qPCR and compared to Gapdh. Mean and standard deviation (M + SD), n = 5. *P < 0.05, **P < 0.01, ns—not significant. (G) Myc-PRH and Myc-PRHΔC EE protein levels in the experiment described in (F) were determined by western blotting. Lamin A/C was used as a control for loading.

Figure 7.

The transdominant negative activity of PRHΔC requires binding to TLE co-repressor proteins. (A) K562 cells were co-transfected with expression vectors for FLAG-TLE1 and Myc-PRH or FLAG-TLE1 and Myc-PRHΔC EE and nuclear extracts prepared for co-immunoprecipitation. The top panel shows a western blot for FLAG-TLE1 in the nuclear extract (1 and 4) and in the same extract after immunoprecipitation with the Myc9E10 antibody (2 and 5) or control rabbit IgG antibody (3 and 6). The blot was striped and reprobed with the Myc9E10 antibody to confirm expression of Myc-PRH and Myc-PRHΔC EE (bottom panel). The secondary antibody also picks up the mouse IgG light chain (IgG LC). (B) K562 cells were transfected with an expression vector for FLAG tagged TLE1 and nuclear extracts prepared for co-immunoprecipitation. The bottom panel shows a western blot for endogenous pPRH in the nuclear extract (1) and after immunoprecipitation with the FLAG antibody (2) or control mouse IgG antibody (3). (C) Vegfr-1 mRNA levels in K562 cells 48 h post-transfection with plasmids expressing Myc-PRH (5 µg) alone, Myc-PRH (5 µg) and Myc-PRHΔC EE (5 µg) or and Myc-PRH and Myc-PRHΔC EE F32E (5 µg). mRNA levels were determined by qPCR and compared to Gapdh. Mean and standard deviation (M + SD), n = 3. *P < 0.05, (D) Cell extracts from (C) were used to determine the expression levels of Myc-PRHΔC EE and Myc-PRHΔC EE F32E using western blotting. The blot was stripped and reprobed for Lamin A/C as a loading control.