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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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Related in: MedlinePlus

Plk1 binds to STARD9-MD, phosphorylates it at serine 312, and regulates its degradation. (A) Immunoprecipitation of LAP-tagged STARD9-MD wild type and S317A mutant from G1/S or mitotic extracts. Eluates were immunoblotted for the indicated proteins. Note that Plk1 only coimmunoprecipitates with mitotic phosphorylated wild-type STARD9-MD and not the nonphosphorylated S317A STARD9-MD mutant. NI indicates the noninduced control. (B) Plk1 phosphorylates STARD9-MD at serine 312. In vitro phosphorylation assays were carried out with recombinant wild type or serine-to-alanine mutant STARD9-MD. The transfer of the [γ-32P]phosphate group onto STARD9-MD was monitored by Western blot and radiometric analyses. (C) Plk1 regulates STARD9-MD protein levels in vivo. The LAP-tagged STARD9-MD cell line was synchronized in G1/S and released into nocodazole-containing media in the presence or absence of BI2536 or GSK461364 Plk1 inhibitors, mitotic cells were harvested 20 h postrelease, protein extracts were analyzed by immunoblotting for the indicated proteins, and the levels of STARD9-MD were quantified for each condition. Data represent the average ± SD of three independent experiments. (D) siRNA knockdown of endogenous Plk1 protein in LAP-tagged STARD9-MD wild-type and S312A mutant cell lines. Cells were synchronized in early mitosis with nocodazole for 16 h and released, and samples were collected at the indicated time points and immunoblotted with the indicated antibodies. CTRL indicates control siRNA. (E) The LAP-tagged STARD9-MD wild-type and S312A cell lines were transfected with HA-Plk1 or HA-Plk1-KD overexpression vectors, cells were arrested in mitosis with nocodazole for 16 h and released into fresh medium, and extracts were prepared at the indicated time points and immunoblotted with the indicated antibodies.
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Figure 5: Plk1 binds to STARD9-MD, phosphorylates it at serine 312, and regulates its degradation. (A) Immunoprecipitation of LAP-tagged STARD9-MD wild type and S317A mutant from G1/S or mitotic extracts. Eluates were immunoblotted for the indicated proteins. Note that Plk1 only coimmunoprecipitates with mitotic phosphorylated wild-type STARD9-MD and not the nonphosphorylated S317A STARD9-MD mutant. NI indicates the noninduced control. (B) Plk1 phosphorylates STARD9-MD at serine 312. In vitro phosphorylation assays were carried out with recombinant wild type or serine-to-alanine mutant STARD9-MD. The transfer of the [γ-32P]phosphate group onto STARD9-MD was monitored by Western blot and radiometric analyses. (C) Plk1 regulates STARD9-MD protein levels in vivo. The LAP-tagged STARD9-MD cell line was synchronized in G1/S and released into nocodazole-containing media in the presence or absence of BI2536 or GSK461364 Plk1 inhibitors, mitotic cells were harvested 20 h postrelease, protein extracts were analyzed by immunoblotting for the indicated proteins, and the levels of STARD9-MD were quantified for each condition. Data represent the average ± SD of three independent experiments. (D) siRNA knockdown of endogenous Plk1 protein in LAP-tagged STARD9-MD wild-type and S312A mutant cell lines. Cells were synchronized in early mitosis with nocodazole for 16 h and released, and samples were collected at the indicated time points and immunoblotted with the indicated antibodies. CTRL indicates control siRNA. (E) The LAP-tagged STARD9-MD wild-type and S312A cell lines were transfected with HA-Plk1 or HA-Plk1-KD overexpression vectors, cells were arrested in mitosis with nocodazole for 16 h and released into fresh medium, and extracts were prepared at the indicated time points and immunoblotted with the indicated antibodies.

Mentions: Given that STARD9-MD harbored Plkl binding sites that were phosphorylated in mitosis (S304pS305 and S316pS317; Figure 3, C–E), we asked whether Plk1 bound to STARD9-MD and was able to phosphorylate it. LAP-tagged STARD9-MD wild type or S317A was immunoprecipitated from G1/S or mitotic cells, and eluates were immunoblotted for Plk1. Indeed, endogenous Plk1 bound preferentially to the mitotic phosphorylated STARD9-MD in comparison to the G1/S nonphosphorylated STARD9-MD (Figure 5A). Furthermore, endogenous Plk1 was unable to bind the nondegradable/nonphosphorylated S317A STARD9-MD mutant (Figure 5A). Consistently, overexpressed hemagglutinin (HA)-Plk1-KD (kinase-dead form, binds to substrates but does not phosphorylate them) coimmunoprecipitated with STARD9-MD from mitotic cells (Supplemental Figure S6). The interaction between Plk1 and phosphorylated STARD9-MD was also assessed by in vitro binding assays in which in vitro translated HA-Plk1 or HA-Plk1-KD both immunoprecipitated preferentially with recombinant phosphorylated (incubated with mitotic extracts) GST-STARD9-MD compared with nonphosphorylated (not incubated with mitotic extracts) STARD9-MD and control GST (Supplemental Figure S6). These results indicated that Plk1 was binding to phosphorylated STARD9-MD. Next we asked whether Plk1 could directly phosphorylate STARD9-MD. In vitro phosphorylation assays were carried out with recombinant wild type or serine-to-alanine STARD9-MD mutants and [γ-32P]ATP in the presence or absence of recombinant Plk1. The transfer of the [γ-32P]phosphate group onto STARD9-MD was then monitored by Western blot and radiometric analyses. In this assay, Plk1 was able to phosphorylate wild type and S304A, S305A, S316A, and S317A single mutants, although S304A, S305A, and S316A had a minimal reduction in phosphorylation compared with wild type. However, only minimal phosphorylation was observed with the S312A single mutant and the double and triple mutants that also contained the S312A mutation (Figure 5B). These results indicated that Plk1 was phosphorylating S312 and that phosphorylation at other serines was likely due to other mitotic kinases. Finally, we asked whether Plk1 regulated STARD9-MD protein levels in vivo. Treatment of cells with the Plk1 inhibitor BI2536 or GSK461364 led to the stabilization of STARD9-MD in mitosis (a representative immunoblot is shown in Figure 5C). Consistently, depletion of Plk1 in LAP-tagged STARD9-MD wild-type cells, which had been synchronized in early mitosis with nocodazole and released into the cell cycle, inhibited the degradation of STARD9-MD (Figure 5D). To assess further whether Plk1 phosphorylation was regulating the degradation of STARD9-MD, we transfected the LAP-tagged STARD9-MD cell line with HA-Plk1 or HA-Plk1-KD overexpression vectors and monitored the stability of STARD9-MD by immunoblot analysis during release from early mitosis. Overexpression of HA-Plk1 led to a decrease in STARD9-MD protein levels, whereas overexpression of HA-Plk1-KD (binds and inhibits phosphorylation of substrates) led to a stabilization of STARD9-MD (Figure 5E). On the other hand, the depletion of Plk1 (Figure 5D) or overexpression of Plk1 or Plk1-KD (Figure 5E) did not change the protein levels of S312A STARD9-MD mutant, indicating that phosphorylation of S312 played an important role in the modulation of STARD9-MD protein levels. Together these results indicated that Plk1 was able to bind to phosphorylated STARD9-MD and was responsible for phosphorylating it at serine 312 and that this phosphorylation event was critical for regulating STARD9-MD protein stability.


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)

Plk1 binds to STARD9-MD, phosphorylates it at serine 312, and regulates its degradation. (A) Immunoprecipitation of LAP-tagged STARD9-MD wild type and S317A mutant from G1/S or mitotic extracts. Eluates were immunoblotted for the indicated proteins. Note that Plk1 only coimmunoprecipitates with mitotic phosphorylated wild-type STARD9-MD and not the nonphosphorylated S317A STARD9-MD mutant. NI indicates the noninduced control. (B) Plk1 phosphorylates STARD9-MD at serine 312. In vitro phosphorylation assays were carried out with recombinant wild type or serine-to-alanine mutant STARD9-MD. The transfer of the [γ-32P]phosphate group onto STARD9-MD was monitored by Western blot and radiometric analyses. (C) Plk1 regulates STARD9-MD protein levels in vivo. The LAP-tagged STARD9-MD cell line was synchronized in G1/S and released into nocodazole-containing media in the presence or absence of BI2536 or GSK461364 Plk1 inhibitors, mitotic cells were harvested 20 h postrelease, protein extracts were analyzed by immunoblotting for the indicated proteins, and the levels of STARD9-MD were quantified for each condition. Data represent the average ± SD of three independent experiments. (D) siRNA knockdown of endogenous Plk1 protein in LAP-tagged STARD9-MD wild-type and S312A mutant cell lines. Cells were synchronized in early mitosis with nocodazole for 16 h and released, and samples were collected at the indicated time points and immunoblotted with the indicated antibodies. CTRL indicates control siRNA. (E) The LAP-tagged STARD9-MD wild-type and S312A cell lines were transfected with HA-Plk1 or HA-Plk1-KD overexpression vectors, cells were arrested in mitosis with nocodazole for 16 h and released into fresh medium, and extracts were prepared at the indicated time points and immunoblotted with the indicated antibodies.
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Figure 5: Plk1 binds to STARD9-MD, phosphorylates it at serine 312, and regulates its degradation. (A) Immunoprecipitation of LAP-tagged STARD9-MD wild type and S317A mutant from G1/S or mitotic extracts. Eluates were immunoblotted for the indicated proteins. Note that Plk1 only coimmunoprecipitates with mitotic phosphorylated wild-type STARD9-MD and not the nonphosphorylated S317A STARD9-MD mutant. NI indicates the noninduced control. (B) Plk1 phosphorylates STARD9-MD at serine 312. In vitro phosphorylation assays were carried out with recombinant wild type or serine-to-alanine mutant STARD9-MD. The transfer of the [γ-32P]phosphate group onto STARD9-MD was monitored by Western blot and radiometric analyses. (C) Plk1 regulates STARD9-MD protein levels in vivo. The LAP-tagged STARD9-MD cell line was synchronized in G1/S and released into nocodazole-containing media in the presence or absence of BI2536 or GSK461364 Plk1 inhibitors, mitotic cells were harvested 20 h postrelease, protein extracts were analyzed by immunoblotting for the indicated proteins, and the levels of STARD9-MD were quantified for each condition. Data represent the average ± SD of three independent experiments. (D) siRNA knockdown of endogenous Plk1 protein in LAP-tagged STARD9-MD wild-type and S312A mutant cell lines. Cells were synchronized in early mitosis with nocodazole for 16 h and released, and samples were collected at the indicated time points and immunoblotted with the indicated antibodies. CTRL indicates control siRNA. (E) The LAP-tagged STARD9-MD wild-type and S312A cell lines were transfected with HA-Plk1 or HA-Plk1-KD overexpression vectors, cells were arrested in mitosis with nocodazole for 16 h and released into fresh medium, and extracts were prepared at the indicated time points and immunoblotted with the indicated antibodies.
Mentions: Given that STARD9-MD harbored Plkl binding sites that were phosphorylated in mitosis (S304pS305 and S316pS317; Figure 3, C–E), we asked whether Plk1 bound to STARD9-MD and was able to phosphorylate it. LAP-tagged STARD9-MD wild type or S317A was immunoprecipitated from G1/S or mitotic cells, and eluates were immunoblotted for Plk1. Indeed, endogenous Plk1 bound preferentially to the mitotic phosphorylated STARD9-MD in comparison to the G1/S nonphosphorylated STARD9-MD (Figure 5A). Furthermore, endogenous Plk1 was unable to bind the nondegradable/nonphosphorylated S317A STARD9-MD mutant (Figure 5A). Consistently, overexpressed hemagglutinin (HA)-Plk1-KD (kinase-dead form, binds to substrates but does not phosphorylate them) coimmunoprecipitated with STARD9-MD from mitotic cells (Supplemental Figure S6). The interaction between Plk1 and phosphorylated STARD9-MD was also assessed by in vitro binding assays in which in vitro translated HA-Plk1 or HA-Plk1-KD both immunoprecipitated preferentially with recombinant phosphorylated (incubated with mitotic extracts) GST-STARD9-MD compared with nonphosphorylated (not incubated with mitotic extracts) STARD9-MD and control GST (Supplemental Figure S6). These results indicated that Plk1 was binding to phosphorylated STARD9-MD. Next we asked whether Plk1 could directly phosphorylate STARD9-MD. In vitro phosphorylation assays were carried out with recombinant wild type or serine-to-alanine STARD9-MD mutants and [γ-32P]ATP in the presence or absence of recombinant Plk1. The transfer of the [γ-32P]phosphate group onto STARD9-MD was then monitored by Western blot and radiometric analyses. In this assay, Plk1 was able to phosphorylate wild type and S304A, S305A, S316A, and S317A single mutants, although S304A, S305A, and S316A had a minimal reduction in phosphorylation compared with wild type. However, only minimal phosphorylation was observed with the S312A single mutant and the double and triple mutants that also contained the S312A mutation (Figure 5B). These results indicated that Plk1 was phosphorylating S312 and that phosphorylation at other serines was likely due to other mitotic kinases. Finally, we asked whether Plk1 regulated STARD9-MD protein levels in vivo. Treatment of cells with the Plk1 inhibitor BI2536 or GSK461364 led to the stabilization of STARD9-MD in mitosis (a representative immunoblot is shown in Figure 5C). Consistently, depletion of Plk1 in LAP-tagged STARD9-MD wild-type cells, which had been synchronized in early mitosis with nocodazole and released into the cell cycle, inhibited the degradation of STARD9-MD (Figure 5D). To assess further whether Plk1 phosphorylation was regulating the degradation of STARD9-MD, we transfected the LAP-tagged STARD9-MD cell line with HA-Plk1 or HA-Plk1-KD overexpression vectors and monitored the stability of STARD9-MD by immunoblot analysis during release from early mitosis. Overexpression of HA-Plk1 led to a decrease in STARD9-MD protein levels, whereas overexpression of HA-Plk1-KD (binds and inhibits phosphorylation of substrates) led to a stabilization of STARD9-MD (Figure 5E). On the other hand, the depletion of Plk1 (Figure 5D) or overexpression of Plk1 or Plk1-KD (Figure 5E) did not change the protein levels of S312A STARD9-MD mutant, indicating that phosphorylation of S312 played an important role in the modulation of STARD9-MD protein levels. Together these results indicated that Plk1 was able to bind to phosphorylated STARD9-MD and was responsible for phosphorylating it at serine 312 and that this phosphorylation event was critical for regulating STARD9-MD protein stability.

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