TAZ promotes PC2 degradation through a SCFbeta-Trcp E3 ligase complex - PubMed (original) (raw)

TAZ promotes PC2 degradation through a SCFbeta-Trcp E3 ligase complex

Yu Tian et al. Mol Cell Biol. 2007 Sep.

Abstract

Studies of a TAZ knockout mouse reveal a novel function of the transcriptional regulator TAZ, that is, as a binding partner of the F-box protein beta-Trcp. TAZ-/- mice develop polycystic kidney disease (PKD) and emphysema. The calcium-permeable cation channel protein polycystin 2 (PC2) is overexpressed in kidneys of TAZ-/- mice as a result of decreased degradation via an SCF(beta-Trcp) E3 ubiquitin ligase pathway. Replacements of serines in a phosphodegron motif in TAZ prevent beta-Trcp binding and PC2 degradation. Coexpression of a cytoplasmic fragment of polycystin 1 blocks the PC2-TAZ interaction and prevents TAZ-mediated degradation of PC2. Depletion of TAZ in zebrafish also results in a cystic kidney accompanied by overexpression of PC2. These results establish a common role of TAZ across vertebrate species in a protein degradation pathway regulated by phosphorylation and implicate deficiencies in this pathway in the development of PKD.

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Figures

FIG. 1.

TAZ−/− mice develop PKD and emphysema. (A) Generation of TAZ knockout mice. WW, WW domain; CC, coiled-coil region; E1, E2, and E3, exons 1, 2, and 3. Selective markers pGKneo and pGKtk are shown in shaded boxes. B, BglII; S, SaclI; H, HindIII. (See Fig. S1 in the supplemental material for more detail.) (B) Progeny of +/− × +/− crosses. TAZ−/− mice were born at a lower-than-expected frequency. (C) Littermates from a heterozygous cross: the mouse on the left is −/−, and those on the right are either +/+ or +/−. (D) Gross and microscopic pictures of kidneys of normal (+/+) and affected (−/−) mice (139 days). Kidneys of the TAZ−/− mouse are enlarged with fluid-filled cysts. (E) Relative kidney size and number of cysts in TAZ−/− mice of different ages. (F) Gross and microscopic pictures of lungs of normal (+/+) and affected (−/−) mice (16 days). Lungs of the TAZ−/− mouse show swollen alveoli with breakdown of alveolar walls.

FIG. 2.

TAZ affects levels of PC2 expression without an effect on PKD2 RNA levels. (A) Origins of renal cysts in a 2-week-old TAZ−/− mouse. Panels left to right: H&E staining; staining with fluorescein isothiocyanate-coupled Lotus tetragonolobus agglutinin as a marker of proximal tubules; higher magnification of the area outlined in second panel with arrows indicating staining of cells lining a cyst; staining of the same section with tetramethyl rhodamine isothiocyanate-coupled DBA as a marker of collecting ducts. (B) Expression levels and localization of PC1 and PC2 in mouse kidneys. Magnification, ×400. Upper panels: staining of kidneys of 3-month-old mice with anti-PC1 antibody (sc-10374; Santa Cruz). Panels left to right: +/+ kidney with normal IgG control, +/+ with anti-PC1; −/− with anti-PC1. Middle panels: staining of kidneys of 3-month-old mice with anti-PC2 antibody 5459. Left to right: +/+ control with antibody preadsorbed with GST-PC2 C-terminal cytoplasmic fragment; +/+ anti-PC2; −/− anti-PC2. Lower panels: embryonic kidneys (14.5 days) with anti-PC2 antibody 5459. Left to right: +/+ with normal IgG control; +/+ anti-PC2; −/− anti-PC2. (C and D) Lysates from mouse kidney tissues (C) and cultured BMK cells (D) from +/+, +/−, and −/− mice were blotted with anti-PC1 (sc-25570; Santa Cruz), anti-PC2 antibody (5459), anti-β-tubulin (T0198; Sigma), and anti-TAZ antibodies. Positive controls are lysates from 293 cells transfected with PC2 and TAZ expression constructs. (E and F) Real-time PCR for PKD1 (E) and PKD2 (F) using total RNA extracts from +/+, +/−, and −/− mouse kidneys at different ages. Results are normalized to GAPDH.

FIG. 3.

Downregulation of TAZ in zebrafish embryos results in renal cyst formation and overexpression of PC2. (A and B) Whole mounts of embryos demonstrate pronounced ventral curvature in the TAZ knockdown animal. (C and D) H&E staining showing a pronephric tubule (pt) in the control embryo (C) and corresponding cystic dilatation of the pronephric tubules (asterisk) in the TAZ-depleted embryo (D). (E and F) Staining of saggital sections of the control (E) and TAZ MO (F) with anti-PC2 (antibody 5459) showing elevated PC2 protein levels in the TAZ knockdown mouse.

FIG. 4.

TAZ promotes PC2 turnover. (A) Pulse-chase experiment in [35S]methionine-labeled 293 cells cotransfected with HA-tagged mPKD2 with TAZ or control vector pcDNA3.1(+). Cells were labeled for 6 h, washed, and incubated further in medium with cold methionine. Cell lysates were made at the indicated times, immunoprecipitated with anti-HA antibody (sc-805; Santa Cruz), and separated by SDS-PAGE. Labeled PC2 protein levels were quantitated by autoradiography and scanning. (B) Established mouse kidney cells of the IMCD line were cotransfected with pMXS-puro plasmid along with a TAZ expression construct or control vector pcDNA3.1(+). After puromycin selection, total cell lysates were separated by SDS-PAGE and blotted for endogenous PC2, TAZ, and tubulin. (C) 293 cells were cotransfected with HA-tagged mPKD2 and pEGFP-N1 along with a TAZ expression construct or control vector pcDNA3.1. Cells were treated with MG132 (5 μM) or dimethyl sulfoxide (DMSO) control for 12 h. Western blot assays for PC2, TAZ, EGFP, and ubiquitin were performed. (D) BMK cells from TAZ+/+ mice were treated with lactacystin (5 μM) for 12 h. Cell lysates were made and immunoprecipitation performed under denaturing conditions (1% SDS) using rabbit anti-PC2 antibody 5459. The Western blot for ubiquitin shows that a fraction of PC2 is ubiquitinylated.

FIG. 5.

TAZ interacts with PC2 and promotes its degradation. (A) Schematic of TAZ and the 290-amino-acid C-terminal portion of mouse PC2. (B) 293 cells were transfected with wild-type TAZ or TAZ 14-3-3 binding mutant S89A/S90A along with a GST fusion to the C-terminal portion of PC2 (GST-PC2c). GST pull-down assays and Western blotting show that PC2c pulls down wild-type TAZ. (C) Wild-type and mutant TAZ were cotransfected along with full-length PC2. The Western blot shows that wild-type but not mutant TAZ is able to promote PC2 degradation. (D thru F) PC2-c, GST-TAZ, TAZ coiled-coil region deletion mutant (ΔCC), and PC1 C-terminal cytoplasmic fragment (residues 4066 to 4293; PC1c) were cotransfected into 293 cells as indicated. GST pull-down assays and Western blotting show that GST-TAZ but not the ΔCC mutant interacts with PC2c and that GST-TAZ/PC2c interaction is inhibited by coexpression of PC1c (D). The Western blot shows that the ΔCC TAZ mutant is ineffective in promoting degradation of PC2 (E). HA-tagged PC2, TAZ, PC1c, and EGFP were cotransfected into 293 cells as indicated. The Western blot shows that PC1c stabilizes PC2 and blocks TAZ-mediated PC2 degradation (F). (G) GST-TAZ, HA-tagged PC2c and mutants were cotransfected into 293 cells as indicated. GST pull-down assay and Western blotting show that the C-terminal 5 amino acids of PC2 resembling a PDZ-binding motif is important for the PC2c/GST-TAZ interaction. (H) GST pull down was performed on an extract of 293 cells transfected with cDNAs of GST-mTAZ and full-length mPC2. The Western blot shows full-length PC2 associated with GST-TAZ. (I) Immunoprecipitation was performed on an extract of 293 cells transfected with cDNAs of mTAZ and HA-tagged mPC2. The Western blot shows association of TAZ with HA-PC2.

FIG. 6.

TAZ mediates PC2 turnover as part of an SCFβ-Trcp E3 ligase complex. (A) HA-tagged mPKD2 and TAZ were cotransfected along with dominant negative constructs of Cul1 (CulDN) and Trcp (TrcpΔF) in 293 cells along with EGFP as a transfection control. The Western blot shows that expression of either CulDN or TrcpΔF prevents TAZ-mediated PC2 degradation. p27 and cdc25A were used as positive controls for disruption by Cul1- and β-Trcp-dependent pathways, respectively. (B) TAZ and mPKD2 cDNAs were cotransfected into 293 cells along with a β-Trcp siRNA construct specific for human β-Trcp. The Western blot shows that PC2 degradation is blocked by β-Trcp siRNA. (C) Immunoprecipitation was performed using anti-PC2 and TAZ antibodies and IMCD cell lysates. Western blots show that TAZ and PC2 associate with endogenous Cul1 and β-Trcp. (D) PC2 ubiquitylation was performed using in vitro-translated 35S-labeled PC2 in the presence of TAZ and SCFβ-Trcp E3 ligase complex. Autoradiography shows that PC2 can be ubiquinylated in vitro in the presence of TAZ and SCFβ-Trcp E3 ligase.

FIG. 7.

Serines 306 and 309 of TAZ are important for TAZ-mediated PC2 degradation. Top: schematic showing sequences of two phosphodegron-like motifs in TAZ. (A and B) cDNAs of wild-type TAZ and TAZ mutants containing serine-to-alanine substitutions were cotransfected along with myc-tagged β-Trcp isoforms into 293 cells. Immunoprecipitation and Western blotting show that the S306A and S309A mutants have lost the ability to bind β-Trcp. (C and D) cDNAs of HA-mPC2, EGFP, and TAZ mutants were cotransfected into 293 cells. The Western blot shows that mutants S306A and S309A have lost the ability to promote PC2 degradation. (E) TAZ−/− MEFs were infected with control, wild type, or S309A mutant TAZ retroviruses. Cells were treated with cycloheximide (CHX; 20 μg/ml) at 24 h postinfection and extracted at the indicated times following CHX addition. Introduction of the wild-type but not mutant S309A TAZ increased the rate of PC2 degradation.

FIG. 8.

Model of TAZ-mediated PC2 protein degradation. (A) TAZ uses its C-terminal phosphodegron motif to link PC2 to an SCFβ-Trcp E3 ligase complex. (B) PC2 degradation is regulated by TAZ phosphorylation. Interaction of PC2 with PC1 protects PC2 from TAZ-mediated degradation.

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TAZ promotes PC2 degradation through a SCFbeta-Trcp E3 ligase complex - PubMed (original) (raw)