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A unique insertion in STARD9's motor domain regulates its stability.

Senese S, Cheung K, Lo YC, Gholkar AA, Xia X, Wohlschlegel JA, Torres JZ - Mol. Biol. Cell (2014)

Bottom Line: These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation.Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects.Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095.

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β-TrCP binds to STARD9-MD and regulates its degradation. (A) GST-STARD9-MD (GST-MD) was incubated with or without mitotic extracts and became phosphorylated in mitotic extracts. (B) β-TrCP binds to STARD9-MD in vitro. GST-STARD9-MD was incubated with HA-β-TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (C) GST-STARD9-MD (GST-MD) or the mutant GST-STARD9-MD S317A (GST-MD S317A) was incubated with or without mitotic extracts, and only GST-MD became phosphorylated in mitotic extracts. (D) GST-STARD9-MD or GST-STARD9-MD S317A was incubated with HA-β -TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (E) The LAP-tagged STARD9-MD wild-type cell line was transfected with HA-β-TrCP or HA-β-TrCPΔF overexpression vectors, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (F) The knockdown efficiency of siRNA targeting β-TrCP expression was determined by RT (reverse transcription)-qPCR. Data represent the average ± SD of three independent experiments. (G) The LAP-tagged STARD9-MD wild-type cell line was transfected with control siRNA or siRNA targeting β-TrCP, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (H) STARD9-MD is a substrate of the SCFβ-TrCP ubiquitin ligase. In vitro ubiquitination reactions were carried out with recombinant GST-STARD9-MD, GST-Emi1, or GST-GFP and with or without ubiquitin, E1, E2, Roc1, Skp1, Cul1, β-TrCP, and mitotic extracts. Products were resolved by SDS–PAGE and immunoblotted with anti-ubiquitin and anti-GST antibodies. Higher-molecular-weight bands are indicative of ubiquitination.
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Figure 6: β-TrCP binds to STARD9-MD and regulates its degradation. (A) GST-STARD9-MD (GST-MD) was incubated with or without mitotic extracts and became phosphorylated in mitotic extracts. (B) β-TrCP binds to STARD9-MD in vitro. GST-STARD9-MD was incubated with HA-β-TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (C) GST-STARD9-MD (GST-MD) or the mutant GST-STARD9-MD S317A (GST-MD S317A) was incubated with or without mitotic extracts, and only GST-MD became phosphorylated in mitotic extracts. (D) GST-STARD9-MD or GST-STARD9-MD S317A was incubated with HA-β -TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (E) The LAP-tagged STARD9-MD wild-type cell line was transfected with HA-β-TrCP or HA-β-TrCPΔF overexpression vectors, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (F) The knockdown efficiency of siRNA targeting β-TrCP expression was determined by RT (reverse transcription)-qPCR. Data represent the average ± SD of three independent experiments. (G) The LAP-tagged STARD9-MD wild-type cell line was transfected with control siRNA or siRNA targeting β-TrCP, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (H) STARD9-MD is a substrate of the SCFβ-TrCP ubiquitin ligase. In vitro ubiquitination reactions were carried out with recombinant GST-STARD9-MD, GST-Emi1, or GST-GFP and with or without ubiquitin, E1, E2, Roc1, Skp1, Cul1, β-TrCP, and mitotic extracts. Products were resolved by SDS–PAGE and immunoblotted with anti-ubiquitin and anti-GST antibodies. Higher-molecular-weight bands are indicative of ubiquitination.

Mentions: The binding of β-TrCP to canonical (DpSG(2X)pS) and extended phosphodegrons (DpSG(3X)pS or DpSG(4X)pS) is critical for SCFβ-TrCP substrate ubiquitination and degradation (Margottin et al., 1998; Busino et al., 2003; Lang et al., 2003). Our mass spectrometry and mutagenesis data indicated that serines 312 and 317 within the STARD9-MD DpSGXXSpS motif were phosphorylated in mitosis and required for STARD9-MD degradation. Thus we asked whether β-TrCP was able to interact with STARD9-MD, by performing coimmunoprecipitation assays. In these assays, phosphorylated or nonphosphorylated GST-STARD9-MD was obtained after incubation with or without M-phase–arrested cell extracts (Figure 6A). The two forms of GST-STARD9-MD were incubated with HA-β-TrCP or HA-β-TrCPΔF (dominant-negative mutant is able to bind substrates but not ubiquitinate them) and immunoprecipitated. The association of HA-β-TrCP or HA-β-TrCPΔF with STARD9-MD was then analyzed by immunoblot analysis. HA-β-TrCP and HA-β-TrCPΔF both immunoprecipitated preferentially with phosphorylated GST-STARD9-MD compared with nonphosphorylated GST-STARD9-MD and control GST (Figure 6B and Supplemental Figure S7). Conversely, β-TrCP did not immunoprecipitate with the nonphosphorylatable/nondegradable S317A STARD9-MD mutant (Figure 6, C and D). To determine the importance of β-TrCP in regulating the levels of STARD9-MD in mitosis, we overexpressed HA-β-TrCP or HA-β-TrCPΔF in cells and analyzed their effect on STARD9-MD protein levels during their release from nocodazole. Overexpression of HA-β-TrCP led to a slight reduction in the levels of STARD9-MD compared with control (CTRL) transfections during mitosis (Figure 6E). In contrast, overexpression of HA-β-TrCPΔF led to the stabilization of STARD9-MD in mitosis (Figure 6E). Accordingly, siRNA-mediated RNA interference targeting endogenous β-TrCP resulted in the stabilization of STARD9-MD (Figure 6, F and G) during mitosis. These results indicated that β-TrCP bound to phosphorylated STARD9-MD and that SCFβ-TrCP was involved in the degradation of STARD9-MD in a cell cycle–dependent manner. To further test this, we asked whether STARD9-MD was an SCFβ-TrCP substrate in an in vitro reconstituted ubiquitination assay (Laney and Hochstrasser, 2011). For these assays, recombinant GST-STARD9-MD was incubated with an ATP-regeneration system, ubiquitin, an E1 ubiquitin- activating enzyme, an E2 ubiquitin-conjugating enzyme, and the SCFβ-TrCP (recombinant Skp1, ±Cul1, ±β-TrCP and Roc1). Reactions were incubated at 30°C for 120 min, and ubiquitination products were resolved by SDS–PAGE and immunoblotted with anti-ubiquitin and anti-GST antibodies. We observed polyubiquitin STARD9-MD as a ladder of increasing molecular weight bands that was largely reduced in the absence of Cul1 or β-TrCP (Figure 6H). In these assays, Emi1 served as a positive control and GFP as a negative control. Together these results indicated that β-TrCP preferentially binds to phosphorylated STARD9-MD and that STARD9-MD is an SCFβ-TrCP substrate.


A unique insertion in STARD9's motor domain regulates its stability.

Senese S, Cheung K, Lo YC, Gholkar AA, Xia X, Wohlschlegel JA, Torres JZ - Mol. Biol. Cell (2014)

β-TrCP binds to STARD9-MD and regulates its degradation. (A) GST-STARD9-MD (GST-MD) was incubated with or without mitotic extracts and became phosphorylated in mitotic extracts. (B) β-TrCP binds to STARD9-MD in vitro. GST-STARD9-MD was incubated with HA-β-TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (C) GST-STARD9-MD (GST-MD) or the mutant GST-STARD9-MD S317A (GST-MD S317A) was incubated with or without mitotic extracts, and only GST-MD became phosphorylated in mitotic extracts. (D) GST-STARD9-MD or GST-STARD9-MD S317A was incubated with HA-β -TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (E) The LAP-tagged STARD9-MD wild-type cell line was transfected with HA-β-TrCP or HA-β-TrCPΔF overexpression vectors, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (F) The knockdown efficiency of siRNA targeting β-TrCP expression was determined by RT (reverse transcription)-qPCR. Data represent the average ± SD of three independent experiments. (G) The LAP-tagged STARD9-MD wild-type cell line was transfected with control siRNA or siRNA targeting β-TrCP, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (H) STARD9-MD is a substrate of the SCFβ-TrCP ubiquitin ligase. In vitro ubiquitination reactions were carried out with recombinant GST-STARD9-MD, GST-Emi1, or GST-GFP and with or without ubiquitin, E1, E2, Roc1, Skp1, Cul1, β-TrCP, and mitotic extracts. Products were resolved by SDS–PAGE and immunoblotted with anti-ubiquitin and anti-GST antibodies. Higher-molecular-weight bands are indicative of ubiquitination.
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Related In: Results  -  Collection

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Figure 6: β-TrCP binds to STARD9-MD and regulates its degradation. (A) GST-STARD9-MD (GST-MD) was incubated with or without mitotic extracts and became phosphorylated in mitotic extracts. (B) β-TrCP binds to STARD9-MD in vitro. GST-STARD9-MD was incubated with HA-β-TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (C) GST-STARD9-MD (GST-MD) or the mutant GST-STARD9-MD S317A (GST-MD S317A) was incubated with or without mitotic extracts, and only GST-MD became phosphorylated in mitotic extracts. (D) GST-STARD9-MD or GST-STARD9-MD S317A was incubated with HA-β -TrCP for 1 h and washed, and the binding was monitored by immunoblot analysis. (E) The LAP-tagged STARD9-MD wild-type cell line was transfected with HA-β-TrCP or HA-β-TrCPΔF overexpression vectors, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (F) The knockdown efficiency of siRNA targeting β-TrCP expression was determined by RT (reverse transcription)-qPCR. Data represent the average ± SD of three independent experiments. (G) The LAP-tagged STARD9-MD wild-type cell line was transfected with control siRNA or siRNA targeting β-TrCP, synchronized in mitosis with nocodazole for 16 h, and released into the cell cycle, and the stability of STARD9-MD was monitored by immunoblot analysis. (H) STARD9-MD is a substrate of the SCFβ-TrCP ubiquitin ligase. In vitro ubiquitination reactions were carried out with recombinant GST-STARD9-MD, GST-Emi1, or GST-GFP and with or without ubiquitin, E1, E2, Roc1, Skp1, Cul1, β-TrCP, and mitotic extracts. Products were resolved by SDS–PAGE and immunoblotted with anti-ubiquitin and anti-GST antibodies. Higher-molecular-weight bands are indicative of ubiquitination.
Mentions: The binding of β-TrCP to canonical (DpSG(2X)pS) and extended phosphodegrons (DpSG(3X)pS or DpSG(4X)pS) is critical for SCFβ-TrCP substrate ubiquitination and degradation (Margottin et al., 1998; Busino et al., 2003; Lang et al., 2003). Our mass spectrometry and mutagenesis data indicated that serines 312 and 317 within the STARD9-MD DpSGXXSpS motif were phosphorylated in mitosis and required for STARD9-MD degradation. Thus we asked whether β-TrCP was able to interact with STARD9-MD, by performing coimmunoprecipitation assays. In these assays, phosphorylated or nonphosphorylated GST-STARD9-MD was obtained after incubation with or without M-phase–arrested cell extracts (Figure 6A). The two forms of GST-STARD9-MD were incubated with HA-β-TrCP or HA-β-TrCPΔF (dominant-negative mutant is able to bind substrates but not ubiquitinate them) and immunoprecipitated. The association of HA-β-TrCP or HA-β-TrCPΔF with STARD9-MD was then analyzed by immunoblot analysis. HA-β-TrCP and HA-β-TrCPΔF both immunoprecipitated preferentially with phosphorylated GST-STARD9-MD compared with nonphosphorylated GST-STARD9-MD and control GST (Figure 6B and Supplemental Figure S7). Conversely, β-TrCP did not immunoprecipitate with the nonphosphorylatable/nondegradable S317A STARD9-MD mutant (Figure 6, C and D). To determine the importance of β-TrCP in regulating the levels of STARD9-MD in mitosis, we overexpressed HA-β-TrCP or HA-β-TrCPΔF in cells and analyzed their effect on STARD9-MD protein levels during their release from nocodazole. Overexpression of HA-β-TrCP led to a slight reduction in the levels of STARD9-MD compared with control (CTRL) transfections during mitosis (Figure 6E). In contrast, overexpression of HA-β-TrCPΔF led to the stabilization of STARD9-MD in mitosis (Figure 6E). Accordingly, siRNA-mediated RNA interference targeting endogenous β-TrCP resulted in the stabilization of STARD9-MD (Figure 6, F and G) during mitosis. These results indicated that β-TrCP bound to phosphorylated STARD9-MD and that SCFβ-TrCP was involved in the degradation of STARD9-MD in a cell cycle–dependent manner. To further test this, we asked whether STARD9-MD was an SCFβ-TrCP substrate in an in vitro reconstituted ubiquitination assay (Laney and Hochstrasser, 2011). For these assays, recombinant GST-STARD9-MD was incubated with an ATP-regeneration system, ubiquitin, an E1 ubiquitin- activating enzyme, an E2 ubiquitin-conjugating enzyme, and the SCFβ-TrCP (recombinant Skp1, ±Cul1, ±β-TrCP and Roc1). Reactions were incubated at 30°C for 120 min, and ubiquitination products were resolved by SDS–PAGE and immunoblotted with anti-ubiquitin and anti-GST antibodies. We observed polyubiquitin STARD9-MD as a ladder of increasing molecular weight bands that was largely reduced in the absence of Cul1 or β-TrCP (Figure 6H). In these assays, Emi1 served as a positive control and GFP as a negative control. Together these results indicated that β-TrCP preferentially binds to phosphorylated STARD9-MD and that STARD9-MD is an SCFβ-TrCP substrate.

Bottom Line: These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation.Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects.Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095.

Show MeSH
Related in: MedlinePlus