PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Oncogene. Author manuscript; available in PMC 2017 September 22.
Published in final edited form as:
PMCID: PMC5086402
NIHMSID: NIHMS824438

Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function

Abstract

Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation, and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long form variant of VHL was often detected as multiple bands by Western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetic and pharmacologic approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein, thereby promoting HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases.

Keywords: VHL, Proteostasis, ccRCC, Phosphorylation, CK2

Introduction

von Hippel Lindau (VHL) disease is an autosomal dominant disorder in which loss of VHL protein function predisposes individuals to benign and malignant tumors, including sporadic and inherited clear cell renal cell carcinoma (ccRCC) (13). The VHL gene is mutated in 80%–90% of cases of ccRCC (4) as an early event during tumorigenesis (5), indicating that loss of VHL function is a key underlying cancer driver. VHL binds to elongins C and B (6) as well as cullin-2 (7), forming an E3 ubiquitin ligase complex that mediates the ubiquitination and subsequent degradation of hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) (8). Stabilization of HIFs due to VHL loss is critical to ccRCC tumorigenesis (9, 10).

VHL has a rapid protein turnover rate (11), suggesting that proteostasis plays a critical role in controlling its functions. Nascent VHL is folded by the eukaryotic type II chaperonin tailless complex polypeptide-1 (TCP-1) ring complex (TRiC), which is also called CCT for chaperonin containing TCP-1 (12, 13). After folding, VHL is then released from TRiC and loaded into the VHL-elongin B-elongin C complex that stabilizes VHL (14). Approximately one-third of all VHL mutations are missense point mutations, generating a full-length but destabilized protein (15, 16). These point mutations, in cases that maintain residual functionality (16, 17), could be stabilized and therefore refunctionalized by genetic or pharmacologic strategies (11). We and others have shown that modulation of the proteostasis of missense point-mutated VHL could serve as a novel approach to treating kidney cancer (11, 18, 19). Therefore, a better understanding of VHL proteostasis is important for identifying new drug targets and developing novel therapeutic approaches targeting VHL deficiency in ccRCC and other cancer types.

VHL is expressed as two variants arising from different translational start sites in the same cDNA, a full-length VHL of 213 amino acids (aa) and a short-form VHL19 lacking the first 53 aa (20, 21). In our previous studies, we observed that full-length VHL, even when expressed from an exogenous cDNA, was always detected as multiple bands, especially after proteasome inhibition (11, 19). Many other studies also showed that VHL was detected as two or more bands (2022). However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are completely unknown.

We hypothesized that the multiple species of VHL are generated by protein cleavage, and the cleavage regulates VHL proteostasis and subsequent protein function. To test our hypothesis, we characterized VHL species using genetic and pharmacologic approaches and showed that VHL was cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2 (CK2)-mediated phosphorylation at the VHL N-terminus was required for the cleavage. Further, inhibition of the cleavage stabilized VHL, thereby consequently promoting HIF downregulation.

Results

VHL is cleaved at its N-terminus

We expressed VHL-wild type (wt) linked to a C-terminal Venus tag in VHL-null 786-0 kidney cancer cells. The VHL fusion was detected as double immunoreactive bands on Western blotting, a major upper band (designated by arrow head) and a minor lower band (arrow) (Fig. 1A). The lower band accumulated markedly after proteasome inhibition by MG132, bortezomib, or carfilzomib (Fig. 1A). Similar lower bands were detected in VHL W117A and L118P mutations that are known to disrupt VHL binding to HIF (Supplementary Fig. 1). We sought to verify that the bands were from the VHL30 variant but not from the short-form VHL19 lacking the first 53 aa (20, 21). The short-form variant, VHL19, was detectable in VHL-wt-Venus cells with a long exposure after proteasome inhibition (designated by a star), which was smaller in molecular weight than the lower band seen following expression of the VHL construct (Fig. 1B). To confirm that the lowest band was VHL19, we mutated the first translational start of VHL-wt-Venus; therefore, VHL was expressed only from the internal translational start. As expected, only a single band at the size of VHL19 was detected (Fig. 1B). The data indicated that the lower band of VHL-wt-Venus was not the VHL19 short variant. Instead, the lower band resulted from post-translational modification(s) of full-length VHL.

Figure 1
VHL is cleaved at the N-terminus. (A) Long form of VHL was expressed as two bands. VHL-null 786-0 cells were infected to stably express VHL-wt-Venus. Cells were cultured in complete medium and treated with indicated inhibitors for 24 hours. MG132 (1 µM); ...

The lower band was also detected with untagged VHL expressed in VHL-null 786-0 or A498 cells after proteasome inhibition, although an extra middle band was also visible between the upper and lower bands (Fig. 1C). We first focused on the upper and lower bands. A similar lower band was detected from endogenous VHL in renal proximal tubule epithelial (RPTEC) and human embryonic kidney (HEK) 293 cells (Fig. 1D), as were the middle smear bands, indicating VHL expressing as multiple bands is generalizable. We further examined tagged and untagged VHL with a different mouse monoclonal VHL antibody (Ig32). Similar upper and lower bands were detected, indicating that the detection of multiple species of VHL was not antibody specific (Supplementary Fig. 2). We estimated the lower band was 2–3 kDa smaller than the upper band based on the locations on Western blots.

Based on these results, we proposed that the lower band was generated from full-length VHL via cleavage at the N-terminus. We then used Edman degradation to analyze N-terminal aa sequences of the upper and lower bands of Venus-tagged VHL purified by GFP-Trap after bortezomib treatment (Supplementary Fig. 3A). The results indicated the first 5 aa of the upper band were PRRAE, corresponding to full-length VHL from its natural N-terminus with the first methionine removed (Supplementary Fig. 3B). Edman degradation did not identify the first aa of the lower band. The results suggested that the second to the fourth aa of the lower band was PEED, consistent with the 23rd tyrosine being the cleavage site (Supplementary Fig. 3B). The size of the 23 aa removed by cleavage at the Tyr23 is about 2.5 kDa, consistent with the finding that the lower band was 2–3 kDa smaller than the upper band. We next made VHL T23 lacking the first 23 aa, which was expressed as a single band at similar size of the lower band (Fig. 1E), further suggesting that VHL was cleaved at tyrosine 23. Similar to untagged or C-terminus Venus-tagged VHL, the lower band was also detected from VHL fused to a small V5 tag at the C-terminus (Fig. 1F). Interestingly, the lower band was not detectable when another small HA tag was fused to the N-terminus of VHL (Fig. 1F), likely because the N-terminal tag blocked VHL cleavage. Taken together, the data indicate that full-length VHL was cleaved at the N-terminus at tyrosine 23, thereby generating a truncated VHL.

Cleavage of VHL leads to proteasomal degradation

We next examined the function of VHL cleavage. The lower band of VHL accumulated markedly after 24 hours of proteasome inhibition (Fig. 1A). The cleaved VHL-wt-Venus lower band started to increase 30 minutes after proteasome inhibition and kept increasing over time (Fig. 2A). In contrast, the upper band maintained constant amounts for the first 6 hours of proteasome inhibition (Fig. 2A). These data suggest that the lower band but not the upper band of VHL was degraded directly through the proteasome. When protein translation was blocked by cycloheximide (CHX), the upper band of VHL started decreasing after 30 minutes. In contrast, the lower band of VHL maintained a constant amount until 180 minutes (Fig. 2B). These data suggest that the upper band but not the lower band of VHL was produced directly through protein synthesis.

Figure 2
Cleavage leads to proteasome degradation of VHL. (A) Changes (over time) in the upper and lower bands of VHL with bortezomib treatment. VHL-wt-Venus 786-0 cells were treated with bortezomib (40 nM) for the indicated time. Cells were lysed in RIPA buffer ...

We then combined bortezomib and CHX to examine the dynamics of the upper and lower bands without VHL synthesis and degradation. About half of the VHL was converted from the upper band to the lower band after 3 hours of bortezomib and CHX treatment (Fig. 2C, lane 3). Most of the VHL was converted to the lower band within 6 hours of treatment with bortezomib and CHX (Fig. 2C, lane 6), and this amount was comparable to that of the upper band before treatment (Fig. 2C, lanes 1 and 6), suggesting that VHL was only cleaved but not degraded within 6 hours of bortezomib-CHX treatment. The amount of the lower bands with or without CHX treatment was comparable (Fig. 2C, lanes 5 and 6), indicating that most of the upper band in lane 5 was newly synthesized in 6 hours. Further, the lower band increased in a nearly linear fashion (Fig. 2A), suggesting that cleavage occurred at a constant rate. Taken together, these data indicate that VHL was first synthesized as the upper band, which was converted into the lower band by cleavage at tyrosine 23 before being directed to the proteasome for degradation. Full length VHL was not degraded directly through the proteasome or other mechanisms before cleavage. The cleavage process, occurring at a constant rate, was a rate-limiting factor for controlling VHL degradation. Once cleaved, VHL was rapidly degraded through the proteasome. Similar cleavage and degradation patterns of the upper and lower bands were observed from untagged VHL (Supplementary Fig. 4 and 5).

VHL cleavage depends on CK2-mediated phosphorylation

Next, we sought to determine the mechanisms that regulate VHL cleavage. Casein kinase 2 (CK2) is a stress-induced kinase that mediates phosphorylation and subsequent degradation of a number of tumor suppressor proteins, including PML (23). CK2 phosphorylates VHL at Ser34, Ser38, and Ser43 at the N-terminus, which is required for the tumor suppressive functions of VHL (24, 25). CK2-mediated phosphorylation was also shown to contribute to VHL degradation (24). However, the mechanism has not been elucidated. We proposed that CK2-mediated phosphorylation regulates the cleavage of VHL and subsequent VHL degradation. A CK2 inhibitor, CX4945, markedly reduced the accumulation of the lower band after proteasome inhibition (Fig. 3A), suggesting that CK2-mediated phosphorylation was required for VHL cleavage. We then knocked down CK2 via siRNA targeting the CK2α subunit. CK2α was efficiently knocked down, and VHL cleavage was largely blocked (Fig. 3B). We then mutated three putative CK2 sites of VHL—Ser33, Ser38, and Ser43—to alanine. As expected, these mutations effectively blocked VHL cleavage (Fig. 3C). Taken together, these data indicate that CK2-mediated phosphorylation of VHL is required for cleavage.

Figure 3
VHL cleavage depends on CK2-mediated phosphorylation. (A) The CK2 inhibitor CX-4945 blocked VHL cleavage at the N-terminus. VHL-wt-Venus 786-0 cells were treated with bortezomib (10 nM or 50 nM), CX-4945 (15 µM), or the two inhibitors combined ...

Chymotrypsin C cleaves VHL

Our mutation and Edman degradation data suggested that tyrosine 23 was a primary cleavage site. We sought to identify the protease(s) that cleave VHL at that site. ExPASy Peptide Cutter (http://web.expasy.org/peptide_cutter/) predicts that chymotrypsin cleaves VHL at 2 sites in the N-terminus, the 8th aa tryptophan and the 23rd aa tyrosine. We first examined a cell-permeable serine protease inhibitor, 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) that inhibits chymotrypsin, trypsin, and other serine proteases (26). AEBSF markedly blocked VHL cleavage (Fig. 4A), suggesting that VHL was likely cleaved by serine protease(s). Further, siRNA knockdown of chymotrypsin C markedly blocked VHL cleavage (Fig. 4B), suggesting that chymotrypsin C is a primary serine protease that cleaves VHL. Protein levels of VHL were elevated with AEBSF treatments or chymotrypsin C knockdown, consistent with the cleavage being required for VHL degradation. We also showed that VHL was cleaved at comparable levels under hypoxia conditions demonstrating that the cleavage is not regulated by oxygen levels (Fig. 4C).

Figure 4
Chymotrypsin C cleaves VHL independent of oxygen levels. (A) A serine protease inhibitor AEBSF blocked VHL cleavage. VHL-wt-Venus 786-0 cells were treated with MG132 (10 µM), AEBSF (100µM), or the two inhibitors combined for 6 hours. Proteins ...

Inhibition of VHL cleavage stabilizes mutant VHL and subsequently downregulates HIF

Restabilization of mutant VHL has been proposed by multiple groups as a novel therapeutic approach to restoring mutant VHL function (11, 18, 19). We sought to determine whether blockage of VHL cleavage via genetic or pharmacologic approaches stabilizes VHL, with a particular focus on mutant VHL. The half-life of the VHL-AAA-Venus protein was markedly longer than that of VHL-wt-Venus (Fig. 5A), indicating that blockage of VHL phosphorylation and subsequent VHL cleavage inhibited proteasome degradation. Similarly, inhibition of CK2 by CX4945 also resulted in a marked increase in VHL levels (Fig. 5B, lanes 2 and 7). As shown in figure 4B and previous studies (11), even low levels of VHL wt could efficiently downregulate HIF2α under normoxic conditions. Therefore, we focused on a patient derived mutation VHL R82P, which is largely deficient in downregulating HIF. CX4945 blocked cleavage, resulting in higher levels of VHL R82P protein (Fig. 5B, lanes 8 and 13). Accordingly, HIF2α protein levels were lower in cells with higher VHL protein levels after CK2 inhibition (Fig. 5B). In contrast, HIF2α protein levels were not affected by CK2 inhibition when VHL was not present in parental 786-0 cells (Fig. 5C), indicating that downregulation of HIF2α in VHL R82P cells was dependent on mutant VHL stabilization. These data indicate that blockage of VHL cleavage via genetic or pharmacologic approaches stabilizes VHL protein and subsequently promotes HIF downregulation.

Figure 5
Inhibition of VHL cleavage stabilizes a naturally-occurring mutant VHL and subsequently downregulates HIF. (A) VHL mutant not phosphorylated by CK2 was more stable than VHL-wt. VHL-wt-Venus 786-0 cells or VHL-AAA-Venus 786-0 cells were treated with CHX ...

Discussion

In this study, we demonstrated that VHL is cleaved at the N-terminus, and this cleavage regulates VHL proteostasis and function. Our data support a model (Fig. 6) that VHL, when routed for degradation, is first cleaved most likely at tyrosine 23 by chymotrypsin C, which is a rate-limiting factor for subsequent VHL degradation, and then directed to the proteasome for rapid degradation. CK2-mediated phosphorylation is required for VHL cleavage by chymotrypsin C. Blockage of VHL cleavage stabilizes VHL protein and subsequently enhances HIF degradation in cases where HIF regulation is compromised.

Figure 6
Schematic model of the effect of VHL cleavage on proteostasis.

Our data support the notion that tyrosine 23 is likely a primary cleavage site. However, more cleavage sites are possible. In untagged VHL, an extra middle band was observed between the upper and lower bands (Fig. 1C). The middle band maintained a pattern similar to that of the lower band until 180 minutes of CHX treatment (Supplementary Fig. 4), suggesting that the middle band was also converted from the upper band. When cells were treated with both bortezomib and CHX, untagged VHL accumulated at the lower band only (Supplementary Fig. 5), suggesting that the middle band was converted into the lower band as well. Therefore, the middle band is likely an intermediate product that is converted during the cleavage process from the upper band to the lower band. Some VHL may have been first cleaved into the middle band and then further cleaved into the lower band. ExPASy Peptide Cutter (http://web.expasy.org/peptide_cutter/) predicts two cleavage sites of VHL by chymotrypsin, the 8th aa tryptophan and the 23rd aa tyrosine, consistent with the cleavage products of the middle and lower bands. The middle band was not observed in Venus-tagged VHL, likely because the middle band was too close to the upper band on blots, or the intermediate products present too briefly to be detected from Venus-tagged VHL.

VHL cleavage was detected in many previous studies (22) 22(20, 21), although it was not further characterized. In some cases, VHL cleavage was not detectable when N-terminal tags were used, likely owing to steric or recognition blockage of VHL cleavage.

We previously proposed that modulation of the proteostasis of missense point-mutated VHL could serve as a novel approach to treating kidney cancer (19) and further showed that proteasome inhibitors can be used to stabilize missense VHL mutations (11). Proteasome inhibitors are only one of many kinds of compounds that may stabilize missense VHL mutations. We previously performed a drug screen and identified multiple compounds that stabilize mutant VHL (19). A recent study also showed that histone deacetylase inhibitors may stabilize mutant VHL (18). We further showed in this study that inhibition of CK2-mediated VHL phosphorylation is another promising approach to stabilizing mutant VHL and downregulating HIF.

CK2 is upregulated as an oncogene in many human cancers including kidney (27), breast (28), and other cancers (29, 30). CK2 inhibitors have been actively tested in multiple cancer types in preclinical and clinical trials (30). CK2 has been shown to regulate HIF1 protein levels (31) and function (32), although the studies suggested the effects were proteasome independent. Our study further demonstrated that CK2 inhibition stabilizes VHL and therefore enhances VHL tumor suppression functions that likely degrade HIF proteins, which provides a novel rationale for CK2 inhibition in cancer therapy.

In summary, we demonstrated that VHL is cleaved at the N-terminus, and this cleavage depends on CK2-mediated phosphorylation. Our study reveals novel mechanisms regulating VHL proteostasis and function, which is of significance for identifying new drug targets and developing new therapeutic approaches to targeting VHL deficiency in ccRCC and potentially other cancer types.

Materials and methods

Cells, reagents, and antibodies

The human RPTEC cell line was obtained from Dr. Kimryn Rathmell (University of North Carolina at Chapel Hill, NC). RCC cells (786-0 and A498) and HEK293T cells were purchased from ATCC (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium from Invitrogen (Carlsbad, CA), supplemented with 10% fetal calf serum from Gibco (Carlsbad, CA). Cell line identity was routinely confirmed via short tandem repeat profiling in the MD Anderson Cancer Center CCSG Characterized Cell Line Core.

MG132 and AEBSF were purchased from Sigma (St. Louis, MO). Bortezomib was purchased from Selleck Chemicals (Houston, TX), and carfilzomib was purchased from Onyx Pharmaceuticals (San Francisco, CA). Three antibodies against VHL were used in this study: one rabbit polyclonal antibody (#2738) from Cell Signaling Technology (Beverly, MA), another rabbit polyclonal antibody (#MBS125962) from MyBioSource (San Diego, CA), and one mouse monoclonal antibody (#OP102, clone Ig33) from EMD Millipore (Billerica, MA). Endogenous VHL was detected using the polyclonal antibody from MyBioSource. Exogenously expressed VHL was detected using the polyclonal antibody from Cell Signaling unless specified otherwise. Anti-HIF2α antibody (#NB100-122) was from Novus Biologicals (Littleton, CO), anti-chymotrypsin C antibody (sc-69253) was from Santa Cruz (Dallas, TX), anti-GAPDH antibody (#AM4300) was from Ambion (Austin, TX), anti-actin antibody (#A5441) was from Sigma-Aldrich (St. Louis, MO), and anti-ERK (#4695) was from Cell Signaling Technology.

Plasmids and stable cell lines

VHL-wt-Venus retroviral expression plasmid was described previously (11, 19). VHL19-Venus for expression of the short form of VHL with a C-terminal Venus tag was constructed from VHL-wt-Venus by disrupting the first ATG translational start with the QuikChange mutagenesis kit from Stratagene (La Jolla, CA) and confirmed by sequencing. VHL-wt for expression of untagged VHL was constructed by cloning VHL cDNA into a retroviral expression vector pBABE-Puro (Addgene, Cambridge, MA, USA, #1764). VHL-R82P for expression of untagged VHL with R82P mutation was constructed from VHL-wt by mutagenesis. VHL-T23-Venus for expression of a truncated VHL lacking the first 23 aa was constructed from VHL-wt-Venus by deleting the cDNA sequence for the first 23 aa of VHL and introducing an ATG translational start. VHL-AAA-Venus was constructed from VHL-wt-Venus by mutating Ser33, Ser38, and Ser43 to alanine via mutagenesis. HA-VHL plasmid was obtained from Addgene (Cambridge, MA, Plasmid #19234). VHL-V5 was constructed from VHL-wt-Venus by replacing the Venus tag with a V5 tag. Retroviruses were prepared, and stable cell lines were generated as previously described (33).

siRNA knockdown

CK2, chymotrypsin C, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cells were transfected according to the manufacturer’s protocol.

Western blotting, GFP-Trap, and Edman degradation

To prepare cell lysates for Western blotting, cells were lysed in RIPA buffer (Tris-HCl, pH 7.4, 50 mM; NaCl, 150 mM; NP-40, 1%; sodium deoxycholate, 0.5%; sodium dodecyl sulfate [SDS], 0.1%) supplemented with protease inhibitor and phosphatase inhibitor cocktail (Pierce, Rockford, IL). Western blotting was performed as previously described (33). Samples were resolved on 10% SDS-polyacrylamide gels. Scanning densitometric values were obtained using ImageJ software (version 1.46r; National Institutes of Health, Bethesda, MD). To prepare proteins for Edman degradation, cells were lysed in RIPA buffer. Venus-tagged VHL proteins were pulled down by the GFP-Trap according to the manufacturer’s protocol (Allele Biotechnology, San Diego, CA) and were then resolved using SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were stained with Coomassie Blue, and target bands were cut for Edman degradation. Edman degradation was performed at the Iowa State University Protein Facility (Ames, IA).

Supplementary Material

Supplementary Figures

Acknowledgments

We thank Dr. Kimryn Rathmell for providing RPTEC cells. This work was supported by a grant from The University of Texas MD Anderson Cancer Center Kidney Cancer Multidisciplinary Research Program (ZD), an MD Anderson Cancer Center Institutional Research Grant (ZD), the NIH grant UO1CA168394 (KLS and GBM), the NIH grant 5 PN2 EY016525-10 (EJ), and NCI CCSG grant to MD Anderson Cancer Center.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

References

1. Lonser RR, Glenn GM, Walther M, Chew EY, Libutti SK, Linehan WM, et al. von Hippel-Lindau disease. Lancet. 2003;361(9374):2059–2067. [PubMed]
2. Jonasch E, Gao J, Rathmell WK. Renal cell carcinoma. BMJ. 2014;349:g4797. [PubMed]
3. Gossage L, Eisen T, Maher ER. VHL, the story of a tumour suppressor gene. Nat Rev Cancer. 2015;15(1):55–64. [PubMed]
4. Nickerson ML, Jaeger E, Shi Y, Durocher JA, Mahurkar S, Zaridze D, et al. Improved identification of von Hippel-Lindau gene alterations in clear cell renal tumors. Clin Cancer Res. 2008;14(15):4726–4734. [PMC free article] [PubMed]
5. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing. New England Journal of Medicine. 2012;366(10):883–892. [PMC free article] [PubMed]
6. Kibel A, Iliopoulos O, DeCaprio JA, Kaelin WG., Jr Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C. Science. 1995;269(5229):1444–1446. [PubMed]
7. Lonergan KM, Iliopoulos O, Ohh M, Kamura T, Conaway RC, Conaway JW, et al. Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2. Mol Cell Biol. 1998;18(2):732–741. [PMC free article] [PubMed]
8. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999;399(6733):271–275. [PubMed]
9. Kondo K, Kim WY, Lechpammer M, Kaelin WG. Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 2003;1:E83. [PMC free article] [PubMed]
10. Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin WG. Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell. 2002;1:237–246. [PubMed]
11. Ding Z, German P, Bai S, Reddy AS, Liu XD, Sun M, et al. Genetic and pharmacological strategies to refunctionalize the von Hippel Lindau R167Q mutant protein. Cancer Res. 2014;74(11):3127–3136. [PMC free article] [PubMed]
12. Feldman DE, Thulasiraman V, Ferreyra RG, Frydman J. Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol Cell. 1999;4(6):1051–1061. [PubMed]
13. Feldman DE, Spiess C, Howard DE, Frydman J. Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol Cell. 2003;12(5):1213–1224. [PubMed]
14. Schoenfeld AR, Davidowitz EJ, Burk RD. Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products. Proc Natl Acad Sci U S A. 2000;97(15):8507–8512. [PubMed]
15. Banks RE, Tirukonda P, Taylor C, Hornigold N, Astuti D, Cohen D, et al. Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer. Cancer Res. 2006;66(4):2000–2011. [PubMed]
16. Rechsteiner MP, von Teichman A, Nowicka A, Sulser T, Schraml P, Moch H. VHL gene mutations and their effects on hypoxia inducible factor HIFalpha: identification of potential driver and passenger mutations. Cancer Res. 2011;71(16):5500–5511. [PubMed]
17. Lee CM, Hickey MM, Sanford CA, McGuire CG, Cowey CL, Simon MC, et al. VHL Type 2B gene mutation moderates HIF dosage in vitro and in vivo. Oncogene. 2009;28(14):1694–1705. [PMC free article] [PubMed]
18. Yang C, Huntoon K, Ksendzovsky A, Zhuang Z, Lonser RR. Proteostasis modulators prolong missense VHL protein activity and halt tumor progression. Cell reports. 2013;3(1):52–59. [PMC free article] [PubMed]
19. Ding Z, German P, Bai S, Feng Z, Gao M, Si W, et al. Agents That Stabilize Mutated von Hippel-Lindau (VHL) Protein: Results of a High-Throughput Screen to Identify Compounds That Modulate VHL Proteostasis. J Biomol Screen. 2012;17(5):572–580. [PMC free article] [PubMed]
20. Schoenfeld A, Davidowitz EJ, Burk RD. A second major native von Hippel-Lindau gene product, initiated from an internal translation start site, functions as a tumor suppressor. Proc Natl Acad Sci U S A. 1998;95(15):8817–8822. [PubMed]
21. Iliopoulos O, Ohh M, Kaelin WG. pVHL19 is a biologically active product of the von Hippel–Lindau gene arising from internal translation initiation. Proceedings of the National Academy of Sciences. 1998;95(20):11661–11666. [PubMed]
22. Duan DR, Humphrey JS, Chen DY, Weng Y, Sukegawa J, Lee S, et al. Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations. Proceedings of the National Academy of Sciences. 1995;92(14):6459–6463. [PubMed]
23. Scaglioni PP, Yung TM, Cai LF, Erdjument-Bromage H, Kaufman AJ, Singh B, et al. A CK2-Dependent Mechanism for Degradation of the PML Tumor Suppressor. Cell. 2006;126(2):269–283. [PubMed]
24. Ampofo E, Kietzmann T, Zimmer A, Jakupovic M, Montenarh M, Gotz C. Phosphorylation of the von Hippel-Lindau protein (VHL) by protein kinase CK2 reduces its protein stability and affects p53 and HIF-1alpha mediated transcription. Int J Biochem Cell Biol. 2010;42(10):1729–1735. [PubMed]
25. Lolkema MP, Gervais ML, Snijckers CM, Hill RP, Giles RH, Voest EE, et al. Tumor suppression by the von Hippel-Lindau protein requires phosphorylation of the acidic domain. J Biol Chem. 2005;280(23):22205–22211. [PubMed]
26. Powers JC, Asgian JL, Ekici ÖD, James KE. Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases. Chemical Reviews. 2002;102(12):4639–4750. [PubMed]
27. Stalter G, Siemer S, Becht E, Ziegler M, Remberger K, Issinger OG. Asymmetric Expression of Protein Kinase CK2 Subunits in Human Kidney Tumors. Biochemical and Biophysical Research Communications. 1994;202(1):141–147. [PubMed]
28. Landesman-Bollag E, Romieu-Mourez R, Song DH, Sonenshein GE, Cardiff RD, Seldin DC. Protein kinase CK2 in mammary gland tumorigenesis. Oncogene. 2001;20(25):3247–3257. [PubMed]
29. Pizzi M, Piazza F, Agostinelli C, Fuligni F, Benvenuti P, Mandato E, et al. Protein kinase CK2 is widely expressed in follicular, Burkitt and diffuse large B-cell lymphomas and propels malignant B-cell growth. 2015 [PMC free article] [PubMed]
30. Montenarh M. Protein kinase CK2 and angiogenesis. Advances in clinical and experimental medicine : official organ Wroclaw Medical University. 2014;23(2):153–158. [PubMed]
31. Guerra B, Rasmussen TDL, Schnitzler A, Jensen HH, Boldyreff BS, Miyata Y, et al. Protein kinase CK2 inhibition is associated with the destabilization of HIF-1α in human cancer cells. Cancer Letters. 2014;356(2):751–761. [PubMed]
32. Mottet D, Ruys SP, Demazy C, Raes M, Michiels C. Role for casein kinase 2 in the regulation of HIF-1 activity. Int J Cancer. 2005;117(5):764–774. [PubMed]
33. Ding Z, Liang J, Lu Y, Yu Q, Songyang Z, Lin SY, et al. A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc Natl Acad Sci U S A. 2006;103(41):15014–15019. [PubMed]