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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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The STARD9 motor domain is modified and degraded in mitosis. (A) The LAP-tagged STARD9-MD cell line was double thymidine blocked (DTB; 0 h) and released into the cell cycle in the presence of nocodazole (NOC; 20 h) ± MG132. Extracts were immunoblotted with indicated antibodies, and the levels of STARD9-MD were quantified (arbitrary units [AU]) for each condition. Data represent the average ± SD of three independent experiments. (B) Same as in A, except that no MG132 was added, and protein extracts were prepared at the indicated time points and analyzed by immunoblotting with the indicated antibodies. The ratio of modified/unmodified STARD9-MD was quantified for each condition as indicated. Data represent the average ± SD of three independent experiments. (C) The LAP-tagged STARD9-MD cell line was synchronized in early mitosis with nocodazole for 16 h and released into fresh medium. Samples were collected each hour and analyzed by immunoblotting with the indicated antibodies. Plk1 was used as a mitotic marker (degraded in early G1 phase). Note that STARD9-MD protein levels decreased during mitosis when Plk1 was present. (D) Recombinant STARD9-MD (GST-MD) was incubated with G1/S or mitotic extracts, and the appearance of the modified form of GST-MD was monitored by immunoblot analysis.
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Figure 2: The STARD9 motor domain is modified and degraded in mitosis. (A) The LAP-tagged STARD9-MD cell line was double thymidine blocked (DTB; 0 h) and released into the cell cycle in the presence of nocodazole (NOC; 20 h) ± MG132. Extracts were immunoblotted with indicated antibodies, and the levels of STARD9-MD were quantified (arbitrary units [AU]) for each condition. Data represent the average ± SD of three independent experiments. (B) Same as in A, except that no MG132 was added, and protein extracts were prepared at the indicated time points and analyzed by immunoblotting with the indicated antibodies. The ratio of modified/unmodified STARD9-MD was quantified for each condition as indicated. Data represent the average ± SD of three independent experiments. (C) The LAP-tagged STARD9-MD cell line was synchronized in early mitosis with nocodazole for 16 h and released into fresh medium. Samples were collected each hour and analyzed by immunoblotting with the indicated antibodies. Plk1 was used as a mitotic marker (degraded in early G1 phase). Note that STARD9-MD protein levels decreased during mitosis when Plk1 was present. (D) Recombinant STARD9-MD (GST-MD) was incubated with G1/S or mitotic extracts, and the appearance of the modified form of GST-MD was monitored by immunoblot analysis.

Mentions: To determine whether STARD9-MD protein levels were cell cycle regulated by the ubiquitin-proteasome system, we generated a doxycycline-inducible localization and affinity purification (LAP = enhanced green fluorescent protein [EGFP]–tobacco etch virus–S-peptide)–tagged STARD9-MD HeLa stable cell line that expresses STARD9-MD from a single specific locus within the genome to monitor STARD9-MD protein levels throughout the cell cycle (Torres et al., 2009). Indeed, STARD9-MD levels remained high in G1/S and mitotic cells in the presence of the proteasome inhibitor MG132 (a representative immunoblot is shown in Figure 2A). However, STARD9-MD protein levels decreased in mitotic cells in the absence of MG132 (a representative immunoblot is shown in Figure 2A). In addition, a higher–molecular weight band corresponding to a modified form of STARD9-MD was apparent in mitotic cells (Figure 2A, arrows). To determine whether this modification was specific for mitosis, we synchronized cells in G1/S and released them into the cell cycle in the presence of nocodazole. Cells were then harvested every 2 h and lysed, and protein samples were analyzed by immunoblot analyses (Figure 2B). STARD9-MD was unmodified in G1/S and became modified at ∼8 h postrelease during mitotic entry (a representative immunoblot is shown in Figure 2B, arrows). To further verify that STARD9-MD levels were decreasing during mitosis, cells synchronized in early mitosis with nocodazole were released, and samples were collected every hour and analyzed by immunoblotting. STARD9-MD protein levels decreased as the cells were released from early mitosis and exited mitosis and increased again as the cells entered G1 (Figure 2C). Similar to the experiments with living cells, recombinant glutathione S-transferase (GST)–STARD9-MD remained unmodified when incubated with G1/S extracts and became modified and degraded when incubated with mitotic extracts (Figure 2D, arrows). These results indicated that STARD9-MD is modified posttranslationally during mitosis and contained the minimal sequences required for STARD9-MD's cell cycle– and proteasome-dependent degradation.


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)

The STARD9 motor domain is modified and degraded in mitosis. (A) The LAP-tagged STARD9-MD cell line was double thymidine blocked (DTB; 0 h) and released into the cell cycle in the presence of nocodazole (NOC; 20 h) ± MG132. Extracts were immunoblotted with indicated antibodies, and the levels of STARD9-MD were quantified (arbitrary units [AU]) for each condition. Data represent the average ± SD of three independent experiments. (B) Same as in A, except that no MG132 was added, and protein extracts were prepared at the indicated time points and analyzed by immunoblotting with the indicated antibodies. The ratio of modified/unmodified STARD9-MD was quantified for each condition as indicated. Data represent the average ± SD of three independent experiments. (C) The LAP-tagged STARD9-MD cell line was synchronized in early mitosis with nocodazole for 16 h and released into fresh medium. Samples were collected each hour and analyzed by immunoblotting with the indicated antibodies. Plk1 was used as a mitotic marker (degraded in early G1 phase). Note that STARD9-MD protein levels decreased during mitosis when Plk1 was present. (D) Recombinant STARD9-MD (GST-MD) was incubated with G1/S or mitotic extracts, and the appearance of the modified form of GST-MD was monitored by immunoblot analysis.
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Figure 2: The STARD9 motor domain is modified and degraded in mitosis. (A) The LAP-tagged STARD9-MD cell line was double thymidine blocked (DTB; 0 h) and released into the cell cycle in the presence of nocodazole (NOC; 20 h) ± MG132. Extracts were immunoblotted with indicated antibodies, and the levels of STARD9-MD were quantified (arbitrary units [AU]) for each condition. Data represent the average ± SD of three independent experiments. (B) Same as in A, except that no MG132 was added, and protein extracts were prepared at the indicated time points and analyzed by immunoblotting with the indicated antibodies. The ratio of modified/unmodified STARD9-MD was quantified for each condition as indicated. Data represent the average ± SD of three independent experiments. (C) The LAP-tagged STARD9-MD cell line was synchronized in early mitosis with nocodazole for 16 h and released into fresh medium. Samples were collected each hour and analyzed by immunoblotting with the indicated antibodies. Plk1 was used as a mitotic marker (degraded in early G1 phase). Note that STARD9-MD protein levels decreased during mitosis when Plk1 was present. (D) Recombinant STARD9-MD (GST-MD) was incubated with G1/S or mitotic extracts, and the appearance of the modified form of GST-MD was monitored by immunoblot analysis.
Mentions: To determine whether STARD9-MD protein levels were cell cycle regulated by the ubiquitin-proteasome system, we generated a doxycycline-inducible localization and affinity purification (LAP = enhanced green fluorescent protein [EGFP]–tobacco etch virus–S-peptide)–tagged STARD9-MD HeLa stable cell line that expresses STARD9-MD from a single specific locus within the genome to monitor STARD9-MD protein levels throughout the cell cycle (Torres et al., 2009). Indeed, STARD9-MD levels remained high in G1/S and mitotic cells in the presence of the proteasome inhibitor MG132 (a representative immunoblot is shown in Figure 2A). However, STARD9-MD protein levels decreased in mitotic cells in the absence of MG132 (a representative immunoblot is shown in Figure 2A). In addition, a higher–molecular weight band corresponding to a modified form of STARD9-MD was apparent in mitotic cells (Figure 2A, arrows). To determine whether this modification was specific for mitosis, we synchronized cells in G1/S and released them into the cell cycle in the presence of nocodazole. Cells were then harvested every 2 h and lysed, and protein samples were analyzed by immunoblot analyses (Figure 2B). STARD9-MD was unmodified in G1/S and became modified at ∼8 h postrelease during mitotic entry (a representative immunoblot is shown in Figure 2B, arrows). To further verify that STARD9-MD levels were decreasing during mitosis, cells synchronized in early mitosis with nocodazole were released, and samples were collected every hour and analyzed by immunoblotting. STARD9-MD protein levels decreased as the cells were released from early mitosis and exited mitosis and increased again as the cells entered G1 (Figure 2C). Similar to the experiments with living cells, recombinant glutathione S-transferase (GST)–STARD9-MD remained unmodified when incubated with G1/S extracts and became modified and degraded when incubated with mitotic extracts (Figure 2D, arrows). These results indicated that STARD9-MD is modified posttranslationally during mitosis and contained the minimal sequences required for STARD9-MD's cell cycle– and proteasome-dependent degradation.

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