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

Abstract

STARD9 is a largely uncharacterized mitotic kinesin and putative cancer target that is critical for regulating pericentriolar material cohesion during bipolar spindle assembly. To begin to understand the mechanisms regulating STARD9 function and their importance to cell division, we took a multidisciplinary approach to define the cis and trans factors that regulate the stability of the STARD9 motor domain. We show that, unlike the other ∼50 mammalian kinesins, STARD9 contains an insertion in loop 12 of its motor domain (MD). Working with the STARD9-MD, we show that it is phosphorylated in mitosis by mitotic kinases that include Plk1. 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.

FIGURE 1:

The STARD9 motor domain loop 12 harbors a unique insertion with Plk1 and β-TrCP binding sites. (A) Schematic of the STARD9 modular domain composition. (B) Molecular modeling of the STARD9 motor domain using the KIF1A motor domain crystal structure (PDB ID 1IA0). Note that loops 7, 11, and 12 are extended. (C) Compared with KIF1A, STARD9 contains a 26–amino acid insertion in loop 12 with putative Plk1 polo-box domain–binding motifs (SS) and a β-TrCP DSGXXS–binding motif. (D) The 26–amino acid insertion and DSGXXS motif are highly conserved among STARD9 mammalian orthologues. (E) Alignment of STARD9, IκBα, claspin, and Emi1 β-TrCP DSGXXS–binding motifs and consensus.

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.

FIGURE 3:

Proteomic analysis of the STARD9 motor domain. (A) STARD9-MD was purified from G1/S (thymidine, 0 h) or mitotic (nocodazole, 12 h) extracts and either treated or not treated with λ phosphatase and analyzed by immunoblot analysis with the indicated antibodies. (B) Recombinant GST-STARD9-MD was incubated with mitotic extracts in the presence or absence of λ phosphatase, and the appearance of the modified form of GST-STARD9-MD was monitored by immunoblot analysis. (C) Tandem affinity purification of LAP-STARD9-MD from G1/S (thymidine, 0 h) or mitotic (nocodazole, 12 h) HeLa cell extracts. Eluates were separated by SDS–PAGE, and protein bands were visualized by silver staining the gel. Immunoblot analysis of these samples was used to verify the identity of STARD9-MD throughout the purification process. The phosphorylated STARD9-MD band present only in mitotic purifications was excised and analyzed by mass spectrometry to map phosphorylation sites. (D) Identified STARD9-MD phosphorylation sites are highlighted with arrows and their amino acid position. Red amino acids were phosphorylated, and yellow amino acids were found to be the most abundantly phosphorylated. (E) The phosphorylation sites were mapped onto the STARD9-MD modeled structure. Nine of 10 phosphorylation sites mapped to the 26–amino acid insertion in the flexible loop 12, and one was outside this region.

FIGURE 4:

Phosphorylation at serines 305, 312, and 317 regulates STARD9-MD protein stability. (A) HeLa stable cell lines expressing STARD9-MD wild type, serine-to-alanine single mutants (S304A, S305A, S312A, S316A, and S317A), double mutants (S312/316A), and triple mutants (S312/316/317) were arrested in G1/S and released into the cell cycle in the presence of nocodazole for 12 or 20 h. Extracts from these time points were immunoblotted to monitor the levels of STARD9-MD and tubulin. (B) HeLa stable cell lines expressing STARD9-MD wild type and serine-to-alanine single mutants (S305A, S312A, and S317A) were arrested in early mitosis with nocodazole for 16 h and treated with cycloheximide during the release. Samples were collected each hour and analyzed by immunoblotting to monitor the levels of STARD9-MD. (C) Molecular modeling of the interaction between β-TrCP and the STARD9-MD extended phosphodegron (DpSGILSpS) peptide.

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.

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.

FIGURE 7:

Increased levels of STARD9-MD lead to spindle assembly and cell division defects. (A) STARD9-MD wild-type and serine-to-alanine mutant stable cell lines were induced to express these proteins. Twenty hours postinduction, cells were fixed and stained for DNA, α-tubulin, and STARD9-MD and imaged by fluorescence microscopy. Bar, 5 μm. (B) Quantification of the fluorescence intensity signal from STARD9-MD wild-type and serine-to-alanine mutant proteins within a 3-μm radius surrounding the centrosome/spindle pole. Data represent the average ± SD of three independent experiments, 20 cells counted for each. *p < 0.05, **p < 0.0005. (C) The percentage of cells with spindle defects was quantified. Data represent the average ± SD of three independent experiments, 100 cells counted for each. *p < 0.05, **p < 0.005, ***p < 0.0005. (D) Live-cell time-lapse microscopy of STARD9-MD wild type and S317A mutant. Top, bright-field (BF) snapshots, and bottom, fluorescence (FL), of STARD-MD localization at the indicated time points in minutes. See Supplemental Movies S1–S4.