ERK1/2 inhibits Cullin 3/SPOP-mediated PrLZ ubiquitination and degradation to modulate prostate cancer progression

Abstract

The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in prostate cancer (PCa), but how SPOP functions as a tumor suppressor and contributes to PCa pathogenesis remains poorly understood. Prostate Leucine Zipper (PrLZ) serves as a prostate-specific and androgen-responsive gene, which plays a pivotal role in the malignant progression of PCa. However, the upstream regulatory mechanism of PrLZ protein stability and its physiological contribution to PCa carcinogenesis remain largely elusive. Here we report that PrLZ can be degraded by SPOP. PrLZ abundance is elevated in SPOP-mutant expressing PCa cell lines and patient specimens. Meanwhile, ERK1/2 might regulate SPOP-mediated PrLZ degradation through phosphorylating PrLZ at Ser40, which blocks the interaction between SPOP and PrLZ. In addition, we identify IL-6 might act as an upstream PrLZ degradation regulator via promoting its phosphorylation by ERK1/2, leading to its impaired recognition by SPOP. Thus, our study reveals a novel SPOP substrate PrLZ which might be controlled by ERK1/2-mediated phosphorylation, thereby facilitating to explore novel drug targets and improve therapeutic strategy for PCa.

Fig. 1. Cullin 1 and Cullin 3 E3 ubiquitin ligases negatively regulate PrLZ protein stability.

a Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from C4-2 cells treated with MG132 (5 and 10 μM) or MLN4924 (0.5 and 1 μM) for 12 h. b IB analysis of WCL derived from 22Rv1 cells under similar treatment condition. c PrLZ binds with Cullin 1 and Cullin 3. IB analysis of WCL and anti-Myc immunoprecipitates (IPs) derived from 293 T cells transfected with Flag-PrLZ and indicated Myc-tagged Cullins. EV, empty vector. d IB analysis of WCL and anti-Flag IPs derived from 293 T cells transfected with Flag-PrLZ and Myc-Cullin 3. EV, empty vector. e IB analysis of WCL derived from C4-2 and 22Rv1 cells stably expressing shCullin 3 or shScr. Scr, Scramble. f IB analysis of WCL derived from C4-2 cells transfected with increasing transfection doses (0.5, 1.5 and 3 μg) of Myc-Cullin 3. EV, empty vector. g IB analysis of WCL derived from 293 T cells transfected with Flag-PrLZ and increasing transfection doses (1.5 and 3 μg) of Myc-Cullin 3. EV, empty vector. h IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, Myc-Cullin 3, and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. i Cullin 3 knockdown cells (shCullin 3), as well as parental C4-2 cells (shScr), were treated with 100 μg/ml cycloheximide (CHX) for the indicated time period before harvesting. Equal amounts of WCL were immunoblotted with the indicated antibodies. j The PrLZ protein abundance in (i) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin.

Fig. 2. SPOP specifically interacts with and promotes PrLZ poly-ubiquitination and degradation.

a PrLZ binds with SPOP. Immunoblot (IB) analysis of WCL and anti-HA immunoprecipitates (IPs) derived from 293 T cells transfected with HA-PrLZ and Flag-tagged BTB domain-containing protein constructs. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. b IB analysis of WCL and anti-SPOP IPs derived from C4-2. Cells were treated with 20 μM MG132 for 6 h before harvesting. c GST pull-down assay revealed the direct interaction between PrLZ and SPOP. The upper panel presents the result of IB by using the antibody against His, and the lower coomassie blue staining showing the gels for purified proteins. d IB analysis of WCL derived from C4-2 cells with SPOP knockout by the CRISPR-Cas9 technology. Parental C4-2 cells are used as the control. e IB analysis of WCL derived from 22Rv1 cells with SPOP knockout by the CRISPR-Cas9 technology. Parental 22Rv1 cells are used as the control. f IB analysis of WCL derived from C4-2 cells transfected with increasing transfection doses (1 and 2 μg) of HA-SPOP. EV, empty vector. g SPOP knockout cells (sgSPOP), as well as parental C4-2 cells (Control), were treated with 100 μg/ml CHX for the indicated time period before harvesting. Equal amounts of WCL were immunoblotted with the indicated antibodies. h The PrLZ protein abundance in (g) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. i IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP, Myc-Cullin 3 and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. j IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP and K-only ubiquitin mutants. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. k IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP and the indicated ubiquitin KR (Lys to Arg) mutants. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. l IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-tagged wild type (WT) and mutanted PrLZ, HA-SPOP, and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. m The growth curve of C4-2 cells with knockdown of SPOP and/or PrLZ. Scr, Scramble. *P < 0.05. n, o Colony formation assays and quantification of C4-2 cells with knockdown of SPOP and/or PrLZ. Scr, Scramble. Error bars represent SEs. *P < 0.05. p, q Representative images and quantification of migrated C4-2 cells with knockdown of SPOP and/or PrLZ. Scr, Scramble. Error bars represent SEs. *P < 0.05.

Fig. 3. PCa-associated SPOP Mutants fail to interact with and promote PrLZ poly-ubiquitination and degradation.

a A schematic diagram representing the SPOP structural domains and PCa-associated mutations for mapping the interaction domains with PrLZ. b Immunoblot (IB) analysis of WCL and anti-Flag immunoprecipitates (IPs) derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, deletion of MATH domain-SPOP constructs, and BTB domain-SPOP constructs. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. WT, wild type. c IB analysis of WCL derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, and deletion of MATH domain-SPOP constructs. EV, empty vector. WT, wild type. d IB analysis of WCL and anti-HA IPs derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, and PCa-associated SPOP mutants. EV, empty vector. WT, wild type. e IB analysis of WCL derived from C4-2 cells stably expressing HA-tagged SPOP WT or PCa-associated SPOP mutants. EV, empty vector. WT, wild type. f IB analysis of WCL derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, and HA-SPOP F102C mutant. Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. EV, empty vector. WT, wild type. g The PrLZ protein abundance in (f) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. h IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP WT, HA-SPOP F102C mutant, and HA-SPOP W131G mutant and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. EV, empty vector. WT, wild type. i-k C4-2 cells stably expressing SPOP-F102C or SPOP-W131G mutants were transfected with shPrLZ or shScr and subcutaneously injected into nude mice to establish xenograft model. Statistical analysis of the tumor volumes which were measured every three days and plotted individually (i). Subcutaneous xenograft tumors formed from different groups were dissected (j). Statistical analysis of the weight of the dissected xenografts tumors (k). n = 6 mice per experimental group, the results indicate the mean ± S.D. *P < 0.05. Scr, Scramble. l Representative images of primary PCa patient samples stained for PrLZ expression by immunohistochemistry. Scale bar, upper 200 μm, lower 100 μm. m Mann–Whitney test analysis of PrLZ expression in primary PCa patient samples harboring SPOP-WT or SPOP-mutations.

Fig. 4. SPOP promotes PrLZ ubiquitination and degradation through interaction with the distinctive N-terminal of PrLZ.

a A schematic diagram representing the PrLZ structural domains for mapping the interaction domains with SPOP. b Immunoblot (IB) analysis of WCL and anti-HA immunoprecipitates (IPs) derived from 293 T cells transfected with GST-SPOP and indicated constructs of HA-PrLZ. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. WT, wild type. c IB analysis of WCL derived from 293 T cells transfected with HA-PrLZ WT, HA-PrLZ animo acid (aa) 46-224 constructs and increasing transfection doses (1 and 3 μg) of Flag-SPOP. WT, wild type. d IB analysis of WCL and anti-Flag IPs derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and deletion of aa 30-42 domain-PrLZ constructs. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. WT, wild type. e IB analysis of WCL derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and deletion of aa 30-42 domain-PrLZ constructs. EV, empty vector. WT, wild type. f IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with HA-SPOP, His-Ub, Flag-PrLZ WT, and deletion of aa 30-42 domain-PrLZ constructs. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. EV, empty vector. WT, wild type. g IB analysis of WCL and anti-Flag IPs derived from 293 T cells transfected with HA-SPOP and indicated mutation constructs of Flag-PrLZ. EV, empty vector. WT, wild type. h IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with HA-SPOP, His-Ub, Flag-PrLZ WT, and Flag-PrLZ S40A mutant. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. EV, empty vector. WT, wild type. i IB analysis of WCL derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and Flag-PrLZ S40A mutant. Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. WT, wild type. j The PrLZ protein abundance in (i) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. k IB analysis of WCL derived from 293 T cells transfected with HA-SPOP and pull-down binding assay using biotinylated peptide.

Fig. 5. ERK1/2-mediated phosphorylation of PrLZ at Ser40 stabilizes PrLZ through disrupting its binding with SPOP.

a Post-translational modifications of PrLZ identified by mass spectrometry (MS). Flag-PrLZ protein derived from 293 T cells were immunoprecipitated with anti-Flag antibody, separated by SDS–PAGE gel and subjected to in-gel digestion for MS. MS/MS spectrum of the PrLZ fragment from S30 to T42, m/z = 672.312 (Z = 2) was shown. b Immunoblot (IB) analysis of WCL and anti-Flag immunoprecipitates (IPs) derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and Flag-PrLZ S40D mutant. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. WT, wild type. c IB analysis of WCL derived from 293 T cells transfected with HA-SPOP, Flag-PrLZ WT, and Flag-PrLZ S40D mutant. Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. WT, wild type. d IB analysis of WCL derived from 293 T cells transfected with GST-SPOP, Flag-PrLZ WT, and indicated kinases constructs. EV, empty vector. e IB analysis of WCL and anti-HA IPs derived from 293 T cells transfected with Flag-PrLZ, HA-ERK1, and HA-ERK2. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. f IB analysis of WCL derived from C4-2 cells treated with SCH772984 (1 and 3 μM) for 24 h. g Sequence alignment of phosphorylation of PrLZ within the SPOP binding domain. h In vitro kinase assays showing that ERK1 phosphorylated recombinant PrLZ at Ser40. i ERK1-mediated phosphorylation of PrLZ hindered its interaction with SPOP in vitro. Autoradiograms showing recovery of ³⁵S-labeled SPOP protein bound to the indicated GST-PrLZ fusion proteins (GST protein as a negative control). IN, input (5% as indicated). j IB analysis of WCL derived from C4-2 and 22Rv1 cells stably expressing shERK1/2 or shScr. Scr, Scramble. k IB analysis of WCL and anti-Flag IPs derived from 293 T cells transfected with Flag-PrLZ and HA-SPOP. 12 h post-transfection, cells were treated with 3 μM SCH772984 for additional 24 h before harvesting. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. l IB analysis of WCL derived from C4-2 cells treated with or without 3 μM SCH772984. Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. WT, wild type. m The PrLZ protein abundance in (l) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. n IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP and His-Ub. 12 h post-transfection, cells were treated with 3 μM SCH772984 for 24 h before harvesting. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. o–q PC-3 cells stably expressing PrLZ-WT or PrLZ-S40A mutant were subcutaneously injected into nude mice with or without SCH772984 treatment (50 mg/kg, daily). Statistical analysis of the tumor volumes which were measured every three days and plotted individually (o). Subcutaneous xenograft tumors from different groups in PC-3 cells were dissected (p). Statistical analysis of the weights of the dissected xenografts tumors (q). n = 6 mice per experimental group, the results indicated the mean ± S.D. *P < 0.05 and ***P < 0.001. NC, non-specific control. WT, wild type. SCH, SCH772984. r Representative images of PCa patient samples stained for PrLZ and p-ERK1/2 expression by immunohistochemistry. Scale bar, left 200 μm, right 100 μm. s Correlation analysis of PrLZ and p-ERK1/2 expression in PCa patient samples. t Mann–Whitney test analysis of PrLZ expression in p-ERK1/2 low and high expression PCa patient samples.

Fig. 6. IL-6 protects PrLZ from degradation through activating ERK1/2.

a Immunoblot (IB) analysis of WCL derived from C4-2 and 22Rv1 cells treated with different concentration of IL-6 (10, 25, 50 and 100 ng/ml) for 48 h. b IB analysis of WCL derived from C4-2 cells treated with IL-6 (50 ng/ml, 48 h) or/and transfection with HA-SPOP. c IB analysis of WCL derived from C4-2 cells treated with IL-6 (50 ng/ml, 48 h) or/and SCH772984 (3 μM). d IB analysis of WCL and anti-Flag immunoprecipitates (IPs) derived from 293 T cells transfected with HA-SPOP and Flag-PrLZ. Cells were treated with IL-6 (50 ng/ml, 48 h) or/and SCH772984 (3 μM). 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. e IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP and His-Ub. Cells were treated with IL-6 (50 ng/ml) or/and SCH772984 (3 μM). Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. f IB analysis of WCL derived from C4-2 cells treated with IL-6 (50 ng/ml) or/and SCH772984 (3 μM). Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. g The PrLZ protein abundance in (f) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. h IB analysis of WCL derived from C4-2 cells transfected with Flag-PrLZ WT and Flag-PrLZ S40A. Cells were treated with IL-6 (50 ng/ml) or/and SCH772984 (3 μM). i, j Colony formation assays and quantitative analysis of C4-2 cells transfected with Flag-PrLZ WT or Flag-PrLZ S40A in the presence or absence of IL-6 (50 ng/ml) or/and SCH772984 (3 μM) treatment. **P < 0.05. k–m C4-2 cells transfected with PrLZ WT or PrLZ S40A were subcutaneously injected into nude mice, which were treated with IL-6 (100 ng per mouse) or/and SCH772984 (SCH, 50 mg/kg, daily). Statistical analysis of the tumor volumes which were measured every three days and plotted individually (k). Subcutaneous xenograft tumors formed from different groups in C4-2 cells were dissected (l). Statistical analysis of the weights of the dissected xenografts tumors (m). n = 6 mice per experimental group, the results indicated the mean ± S.D. *P < 0.05.

Fig. 7. Graphical summary of the proposed mechanism.

A schematic diagram showing the working model for phosphorylation-regulated PrLZ ubiquitination and degradation in modulating PCa progression.