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. 2008 Mar;19(3):899-911.
doi: 10.1091/mbc.e07-07-0631. Epub 2007 Dec 19.

MKKS Is a Centrosome-Shuttling Protein Degraded by Disease-Causing Mutations via CHIP-mediated Ubiquitination

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Free PMC article

MKKS Is a Centrosome-Shuttling Protein Degraded by Disease-Causing Mutations via CHIP-mediated Ubiquitination

Shoshiro Hirayama et al. Mol Biol Cell. .
Free PMC article

Abstract

McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.

Figures

Figure 1.
Figure 1.
Many disease-causing mutants of MKKS are rapidly degraded and prone to aggregation. (A) HEK293 cells were transiently transfected with 0.3 μg of FLAG-tagged wild-type or mutant MKKS expression vectors. Cell lysates were fractionated by centrifugation (15,000 × g; 15 min), and the resulting supernatant (S) and pellet (P) fractions were analyzed by Western blot analysis. Mutants clearly expressing lower levels than wild-type MKKS are boxed with a solid line. NS, nonspecific band. (B) FLAG-tagged MKKS proteins were transiently expressed in HEK293 cells as described in A (top). Alternatively, HEK293 cells stably expressing wild-type or mutant MKKS were produced (bottom). Cells were radiolabeled for 30 min and chased for 3 and 6 h, and cell lysates were analyzed by immunoprecipitation. (C) The radioactivity of the FLAG-MKKS bands (mean and SD) was determined from three transient expression experiments. (D) HEK293 cells were transiently transfected with 0.5 μg of FLAG-tagged wild-type or mutant MKKS expression vectors and analyzed as described in A. Mutants showing greatly enhanced insolubility (P/S ratio equal to or lager than 1.0) are boxed with a dotted line. (E) Classification of disease-causing MKKS mutants according to their enhanced degradation and insolubility characteristics. The upward arrows denote enhanced characteristics relative to the wild-type protein. Mutants failing to localize to the centrosome are underlined (Figure 3). Mutants exhibiting increased insolubility in the presence of MG-132 (Figure 4B) are indicated by asterisks.
Figure 2.
Figure 2.
Wild-type MKKS highly concentrated in the centrosome rapidly shuttles to and from the cytosol. (A) FRAP analysis of MKKS at the centrosome. GFP-MKKS at centrosomes, as indicated by circles, was bleached for 10 ms, and its subsequent movement from the cytosol was monitored. Bar, 5 μm. (B) FLIP experiment was performed by continuous bleaching of a small area (circles) in the cytosol. Bar, 10 μm. (C) Dendra-MKKS at the centrosome was photoconverted from green to red, and movement of the red color emission after the conversion was monitored. Bar, 5 μm. (D) Quantification of the fluorescence intensity of GFP-MKKS during the FRAP experiments (n = 20). CYT and CEN denote cytosol and centrosome, respectively. (E) Fluorescence intensity at centrosomes during FLIP analysis shown in B. (F) Red fluorescence intensity of Dendra-MKKS at the centrosome after photoconversion (n=3) (G and H) MKKS solubility test employing centrifugal fractionation. Transiently expressed untagged MKKS or GFP-MKKS (G) or untagged MKKS expressed in a stable cell line (H) were analyzed by centrifugal fractionation followed by Western blotting by using the MKF-1 anti-MKKS antibody. S, supernatant; P, pellet.
Figure 3.
Figure 3.
Intracellular localization of MKKS mutants. (A and B) HEK293 cells were transiently transfected with FLAG-tagged MKKS expression vectors and stained with the anti-FLAG antibody. Counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI). The mutants shown in B did not localize to the centrosome, whereas those in A did (indicated by arrows). (C) Cells were transfected with the same mutants as in B and treated with 10 μM of the proteasome inhibitor MG-132 for 6 h. Bar, 5 μm. Arrowheads indicate highly concentrated MKKS structures produced by proteasome inhibition.
Figure 4.
Figure 4.
Proteasome inhibition stimulates the formation of insoluble structures by several MKKS mutants. (A) HEK293 cells transfected with MKKS expression vectors (0.3 μg) were treated with 10 μM MG-132. Cell lysates were analyzed by centrifugal fractionation followed by Western blotting. S, supernatant; P, pellet. (B) Western blotting was performed as in A, and the band intensity was quantified (n = 3). Mutants exhibiting pellet/supernatant ratios significantly greater than the wild type are indicated by asterisks (*, p < 0.05; **, p < 0.01). (C) FRAP analysis of MKKS mutants. Cells were transfected with vectors (0.6 μg) expressing GFP-tagged MKKS mutants (Y37C, G345E, or R518H). G345E-expressing cells were treated with MG-132 to inhibit the rapid proteasome-dependent degradation. The centrosome region was bleached for 10 ms, and fluorescence recovery was recorded. (D) Quantification of the fluorescence intensity during FRAP analysis (n = 20). The data of GFP-tagged wild-type MKKS at the centrosome in living and fixed cells are taken from Figure 2D for comparison. (E) Cells transiently expressing FLAG-tagged wild-type or mutant MKKS were lysed in 0.5% TritonX-100/PBS and boiled in the presence of 1% SDS. Cell lysates were analyzed by the filter trap assay using anti-FLAG antibody.
Figure 5.
Figure 5.
Immunofluorescence analysis of HEK293 cells expressing wild-type and mutant MKKS proteins. Cells were transfected with FLAG-tagged wild-type or mutant MKKS. G345E-expressing cells were treated with MG-132 to inhibit rapid degradation. (A–D) Cells were fixed and incubated with mouse (A and C) or rabbit (B and D) anti-FLAG antibodies along with rabbit antibodies against vimentin (A) or γ-tubulin (C), mouse antibody against ubiquitin (B), or rat antibody against HSC70. Cells were then incubated with AlexaFluor 488-conjugated anti-mouse IgG and AlexaFluor 594-conjugated anti-rabbit IgG (A and C), AlexaFluor 594-conjugated anti-rabbit IgG and AlexaFluor 488-conjugated anti-mouse IgG (B), or AlexaFluor 594-conjugated anti-rabbit IgG and AlexaFluor 488-conjugated anti-rat IgG (D). (C) Cells were treated with 20 μM nocodazole. Counterstaining was performed with DAPI except for D. Bar, 10 μm. (E) Double staining of FLAG-MKKS and HSC70 was performed as shown in D, and quantification of merged area in the centrosomal MKKS structure was carried out (n = 10). Asterisks indicate significance of difference (*, p < 0.02; **, p < 0.01).
Figure 6.
Figure 6.
Increased association of CHIP with unstable MKKS mutants and effect of CHIP overexpression on their degradation. (A and B) FLAG-tagged Y37C (unstable and highly insoluble), G345E (unstable and rapidly degraded), or R518H (normal for degradation and solubility) mutants or wild-type MKKS as a control were transiently expressed in HEK293 cells. Cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by Western blotting by using antibodies against molecular chaperones (A) or CHIP (B). Note that the amounts of Y37C and G345E mutants recovered by immunoprecipitation are much lower than those of the R518H mutant and wild-type MKKS due to rapid degradation. (C) Enhanced polyubiquitination of unstable MKKS mutants. Cells were cotransfected with ubiquitin-HA and FLAG-MKKS, and cell lysates were immunoprecipitated with the anti-FLAG antibody. Recovered FLAG-MKKS was analyzed by Western blotting by using antibodies against HA or FLAG. (D) CHIP overexpression reduces the amount of MKKS in both soluble and insoluble fractions. To stimulate MKKS aggregation, transfection efficiency was enhanced by transfecting cells with Effecten instead of FuGENE 6. After cotransfection with FLAG-MKKS (200 ng) and myc-CHIP (0–90 ng), cells lysates were fractionated by centrifugation and analyzed by Western blotting by using antibodies against FLAG or CHIP. (E) Overexpressed and endogenous CHIP proteins were associated with the G345E mutant. Cells were transfected with FLAG-MKKS-G345E as described in D, and FLAG-MKKS-G345E was immunoprecipitated. FLAG-MKKS-G345E and coprecipitated CHIP were analyzed by Western blotting. (F) MKKS synthesis is stimulated by CHIP overexpression. Cells transfected with FLAG-tagged wild-type or mutant MKKS were pulse labeled for 30 min in the presence or absence of CHIP overexpression. Labeled FLAG-MKKS proteins were analyzed by immunoprecipitation with the anti-FLAG antibody. (G and H) Effect of CHIP overexpression on the degradation of the G345E mutant and wild-type MKKS. Cells transiently expressing FLAG-tagged G345E mutant (G) or wild-type MKKS (H) under CHIP overexpression conditions were analyzed by pulse-chase experiments followed by immunoprecipitation. Radioactivity of FLAG-tagged MKKS bands in pulse-chase experiments was determined and normalized against the value of chase at 0 h (n = 3). Asterisks indicate significance of difference (p < 0.05).
Figure 7.
Figure 7.
Polyubiquitination and degradation of the G345E mutant are inhibited by RNAi-mediated knockdown of CHIP. (A) Strong inhibition of MKKS-G345E polyubiquitination by CHIP knockdown. After cotransfection with FLAG-tagged G345E and HA-tagged ubiquitin, cells were treated with CHIP siRNA or nonspecific siRNA as a control. FLAG-G345E was immunoprecipitated with the anti-FLAG antibody and analyzed by Western blotting by using antibodies against HA or FLAG. Expression levels of CHIP were analyzed by Western blotting of total cell extracts. (B) Decreased synthesis of MKKS-G345E by CHIP RNAi treatment. Transiently expressed FLAG-tagged G345E was pulse labeled with [35S]Met for 30 min. Cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by SDS-PAGE. (C and D) Transiently expressed FLAG-tagged G345 mutant (C) and wild-type MKKS (D) were pulse labeled with [35S]Met for 30 min and chased for 1, 2, and 4 h in the presence of CHIP siRNA or nonspecific siRNA as a control. The radiolabeled FLAG-MKKS was immunoprecipitated and analyzed by SDS-PAGE. Radioactivity of FLAG-tagged MKKS bands in pulse-chase experiments was determined and normalized against the value of chase at 0 h (n = 5 in C and n = 4 in D). Asterisks indicate significance of difference (p < 0.02).

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