A unique insertion in STARD9's motor domain regulates its 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.
Affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095.Show MeSH
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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.
Affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095.