br PCa Associated SPOP Mutants Cannot
PCa-Associated SPOP Mutants Cannot Target NANOG for Degradation
SPOP contains a MATH domain that is responsible for the substrate binding and a BTB domain that is required for its inter-action with Cul3 (Zhuang et al., 2009) (Figure 2A). Next, we deter-mined the molecular basis of the interaction between NANOG and SPOP. Our data showed that WT SPOP directly interacted with NANOG, whereas the absence of the MATH domain completely abrogated the binding in vitro and in vivo (Figures 2B and S2A). While deletion of BTB domain in SPOP also reduced the binding to its substrate, the plausible explanation is that the dimerization of SPOP through its BTB domain is required for sufficient interaction with substrates (Marzahn
Figure 2. PCa-Associated SPOP Mutants Are Defective in Promoting NANOG Ubiquitination and Degradation
(A) Schematic diagram of SPOP domains and PCa-associated mutations.
(B) HA-NANOG and indicated FLAG-tag SPOP plasmids were co-expressed in HEK293T cells. Cell lysates were prepared for coIP and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting. (C) FLAG-NANOG and HA-SPOP or mutants were co-expressed in HEK293T cells; protein levels of NANOG and SPOP were analyzed by WB.
(D) FLAG-NANOG, HA-SPOP, or mutants and His-ubiquitin were co-expressed in HEK293T cells. After treatment with MG132 (10 mM) for 6 hr, the Ni-NTA ubiquitination assay was performed and analyzed by WB.
(E) FLAG-NANOG and indicated HA-tag SPOP plasmids were co-expressed in HEK293T cells. Cell lysates were prepared for coIP and WB. Cells were treated with MG132 (10 mM) for 6 hr before harvesting.
(F) FLAG-NANOG was co-expressed in HEK293T SYBR Safe DNA Gel Stain with indicated HA-tag SPOP plasmids. After treating cells with CHX (10 mg/mL) for indicated time intervals, protein levels of NANOG and SPOP were analyzed by WB.
(G) The NANOG protein abundance in (F) was quantified by ImageJ and plotted as indicated.
(H) FLAG-NANOG, HA-SPOP or mutants and His-ubiquitin were co-expressed in HEK293T cells. After treatment with MG132 (10 mM) for 6 hr, the Ni-NTA ubiquitination assay was performed and analyzed by WB.
(I) DU145 cells were infected with lentivirus expressed SPOP or F133L mutants; then the cells were treated with MG132 (10 mM) for 6 hr, cell lysate was IP with anti-NANOG antibody, and the ubiquitination of NANOG was detected by WB using the anti-ubiquitin (Ub) antibody.
(J) Representative sphere images from each condition of DU145 cells. Scale bar, 200 mm.
(L) Representative sphere images from each condition of DU145 cells. Scale bar, 200 mm.
(M) Indicated lentivirus infected DU145 tumor spheres were dissociated, and equal numbers of cells were passaged for three generations. Spheres counts are normalized to the first-generation scrambled shRNA spheres. Data are means ± SEM (n = 3). *p < 0.05, **p < 0.01 versus vector (Student’s t test).
(N) AP staining after 48 hr SPOP or SPOP mutant overexpression in E14 mESCs cells (left) and the expression of SPOP or its mutants and NANOG protein are shown (right).
(P) Immunofluorescent staining analysis for lentivirus expressed SPOP or mutants (green) and endogenous NANOG (red) in E14 mESCs cells. Scale bar, 100 mm.
et al., 2016; Zhang et al., 2014b). Furthermore, our data showed that deletion of either the MATH domain or BTB domain abol-ished the SPOP-mediated NANOG degradation and ubiquitina-tion (Figures 2C and 2D), while overexpression of SPOP, but not its deletion mutants (DBTB or DMATH) in E14 mESCs, led to a loss of mESC morphology and reduced AP staining (Figures S2B–S2D).
Importantly, various somatic mutations have been identified in the MATH domain of SPOP in PCa tissues. Notably, these PCa-associated SPOP mutations, such as Y87C/N, F102C, and F133L/V, mainly occur on the amino acid residues located on the surface of the substrate-binding pocket of the MATH domain and thus lose the ability to bind and degrade its sub-strates (Barbieri et al., 2012; Blattner et al., 2014; Zhuang et al., 2009). Our data indicate that these PCa-associated SPOP mutants are unable to interact with NANOG (Figure 2E). Consistently, ectopic expression of these PCa-associated SPOP mutants failed to degrade NANOG (Figures 2F and 2G). Moreover, these mutants were incapable of promoting NANOG ubiquitination (Figures 2H and 2I). Functionally, the mutants (Y87C and F133L) failed to inhibit the sphere formation of PCa cells (Figures 2J and 2K). Besides, our data showed that cells ectopically expressing NANOG exhibited a great potential to form more and larger spheres than control samples, while such an effect was inhibited by WT-SPOP, but not its F133L mutant, in both DU145 and 22RV1 cells for three consecutive generations (Figures 2L, 2M, S2E, and S2F). Furthermore, expression of WT-SPOP, but not its mutants including Y87C and F133L, impaired the stemness of mESCs (Figures 2N and 2P).