Phosphorylation-Dependent Activation of the ESCRT Function of ALIX in Cytokinetic Abscission and Retroviral Budding

Abstract

The modular adaptor protein ALIX is a key player in multiple ESCRT-III-mediated membrane remodeling processes. ALIX is normally present in a closed conformation due to an intramolecular interaction that renders ALIX unable to perform its ESCRT functions. Here we demonstrate that M phase-specific phosphorylation of the intramolecular interaction site within the proline-rich domain (PRD) of ALIX transforms cytosolic ALIX from closed to open conformation. Defining the role of this mechanism of ALIX regulation in three classical ESCRT-mediated processes revealed that phosphorylation of the intramolecular interaction site in the PRD is required for ALIX to function in cytokinetic abscission and retroviral budding, but not in multivesicular body sorting of activated epidermal growth factor receptor. Thus, phosphorylation of the intramolecular interaction site in the PRD is one of the major mechanisms that activates the ESCRT function of ALIX.

Figure 1. Cytosolic ALIX changes from closed to open conformation during M phase induction

(A) Schematic illustration of the closed and open conformations of ALIX. (B) IB of IE and ME with MPM-2 and 3A9 after Ponceau S (Ponc) staining. (C-E) IP of IE and ME with the indicated antibodies, followed by IB with the indicated antibodies. (F) Incubation of IE and ME with GST or GST-Src, followed by IB of bound proteins with the indicated antibodies. (G) IB of IOE or MEE with MPM-2, and IP of IOE or MEE with the indicated antibodies, followed by IB with 1A3. (H) IB of IE, ME and CIP-treated ME with MPM-2. (I) ME was first incubated with GST or GST-CHMP4b at 4°C for 2 h, and then treated with CIP. IE, ME and the two samples of differently treated ME were immunoprecipitated with the indicated antibodies, followed by IB with the indicated antibodies. See also Figure S1.

Figure 2. MEE treatment of ALIX_nPRD inhibits its interaction with ALIX_Bro1

(A) Schematic illustration of the regions of ALIX that comprise the three structural domains and the ALIX fragments used in this study. (B) GST-ALIX_1-746 was phosphorylated with IOE, MEE, or MEE plus CIP, and end products were immunoprecipitated with the indicated antibodies, followed by IB with α-GST. (C) GST or GST-ALIX_1-746 was phosphorylated with IOE or MEE, and then absorbed onto GSH beads. The washed beads were used to pull down FLAG-CHMP4b in IE. IB of input and bound proteins with the indicated antibodies. (D) Left: experimental flowchart. Middle: IB of input myc-ALIX_nPRD; the short line and the asterisk indicate the start and shift positions of myc-ALIX_nPRD, respectively. Right: IB of bound proteins. (E) Phosphorylation of GST or GST-ALIX_Bro1 with IOE, MEE, or MEE plus CIP, followed by IB of washed proteins with αphosphotyrosine (p-Tyr). (F) Washed GST or GST-ALIX_Bro1 proteins in (E) were tested for interaction with myc-ALIX_nPRD by GST pull-down (n = 3 ± SD).

Figure 3. The S718-S721 phosphorylation inhibits the ALIX_nPRD interaction with ALIX_Bro1

(A) Sequence alignment of the ALIX nPRD with the Xp95 nPRD, and indication of the conserved S/T sites with asterisks. (B&C) Left: IB of mock treated or MEE phosphorylated GST-ALIX_nPRD with #4381 (B) or αpS2 (C). Right: dIP of mock treated or MEE phosphorylated myc-ALIX_nPRD with #4381 (B) or αpS2 (C), followed by IB with αmyc. (D) GST or GST-ALIX_Bro1 pull-down of the indicated forms of myc-ALIX_nPRD after their treatment with IOE, MEE, or MEE plus CIP, followed by IB with the indicated antibodies. (E) Pull-down of the indicated forms of myc-ALIX_nPRD with GST or GST-ALIX_Bro1, followed by IB with the indicated antibodies. (F) GST-ALIX_nPRD was mock treated or phosphorylated at 22°C for 1 h with MEE in the presence or absence of CID755673 (CID), BI-2536 (BI), both inhibitors (C+B) or staurosporine (ST). After the substrate was immobilized onto GSH beads and washed, bound proteins were probed with αpS2 to determine the level of the S718-S721 phosphorylation (n = 3 ± SD). (G) Phosphorylation of GST-ALIX_nPRD with HA-Plx1, followed by IB of the substrate with αpS2.

Figure 4. The S718-S721 phosphorylation relieves the intramolecular interaction of ALIX in mitotic cells

(A) Left: IB of dIE and dME with MPM2. Middle: dIP with #4381, followed by IB with 3A9. Right: dIP with 3A9, followed by IB with 3A9 and αpS2. (B) dIP of IE and ME from cells ectopically expressing WT or S2A GFP-ALIX with #4381, followed by IB with αGFP. (C) dIP of the IE and ME samples in (B) with αGFP, followed by IB with αGFP and αpS2. (D) Left: IB of IE and ME from cells ectopically expressing indicated forms of GFP-ALIX with the indicated antibodies. Right: IP of the IE or ME samples with IgG or the indicated α-ALIX, followed by IB with the indicated antibodies. (E) IP of ME from cells ectopically expressing the indicated forms of GFP-ALIX with IgG or the indicated α-ALIX, followed by IB of input proteins and immunocomplexes with αGFP. (F) IP of IE from cells ectopically coexpressing FLAG-TSG101 and GFP or the indicated forms of GFP-ALIX with αFLAG in the presence of 1% Triton X-100, followed by IB of input proteins and immunocomplexes with the indicated antibodies. (G) Left: experimental flowchart for cell treatments. Middle: IB of IE and ME from collected cells with MPM2. Right: determination of the S718-S721 phosphorylation by dIP with 3A9, followed by IB with 3A9 and αpS2 (n = 3 ± SD). See also Figure S2.

Figure 5. The S718-S721 phosphorylation is required for ALIX to function in cytokinetic abscission

(A) HeLa cells were transfected with the indicated agents and cultured as diagrammed, followed by IB of cell lysates with the indicated antibodies. Fixed cells were stained with αtubulin and αGFP, and GFP-mCherry double positive cells were scored for the midbody localization of mCh-CHMP4b (n = 3 ± SD). Solid and hollow arrowheads in the representative images indicate the presence or absence of mCh-CHMP4b at the midbody, respectively. Enlargements (3×) depict the midbody area; scale bar: 50 μm. (B) HeLa cells were transfected with the indicated agents and cultured as done in (A), followed by IB of cell lysates with the indicated antibodies. Fixed cells were stained with αtubulin and αGFP, and GFP positive cells were scored for midbody-stage or multinucleation (n = 3 ± SD). Solid and hollow arrows in representative images indicate GFP positive mononucleated and multinucleated cells, respectively; hollow arrowheads indicate midbodies between GFP positive cells; and the asterisks indicate the midbody localization of GFP-ALIX*. Scale bar: 50 μm. See also Figure S3.

Figure 6. The S718-S721 phosphorylation is required for ALIX to function in EIAV budding

(A) Incubation of GST or GST-p9 with IE or ME, followed by IB of input and bound proteins with the indicated antibodies. (B) ME was first incubated with GST or GST-p9 at 4°C for 2 h, and then treated with CIP. IE, ME, and the two samples of differently treated ME were immunoprecipitated with IgG or 1A3, followed by IB with the indicated antibodies. (C) Left: experimental flowchart. Middle: IP of cell lysates with the indicated antibodies, followed by IB with αGFP. Right: IB of cell lysates with the indicated antibodies. (D-F) HEK293 cells were processed as diagrammed. IB of VLPs and cell lysates with the indicated antibodies. Relative levels of the VLP production were determined (n = 3 ± SD). See also Figure S4.

Figure 7. The S718-S721 phosphorylation does not affect the function of ALIX in MVB sorting of activated EGFR

(A) HEK293 cells ectopically expressing indicated forms of GFP-ALIX were mock-treated or stimulated with EGF for 1 h, and the average percentage of each GFP-ALIX in the M fraction was determined by membrane flotation centrifugation of the PNS (n = 3 ± SD). (B-F) Left: HEK293 cells were processed as diagrammed, and cell lysates were immunoblotted with the indicated antibodies. Right: cells were stimulated with EGF for 30 min, and assayed for MVB sorting of activated EGFR by the proteinase K protection assay (n = 2 ± data range). (C&D) Cells were stimulated with EGF for indicated minutes, and cell lysates were immunoblotted with the indicated antibodies. The relative levels of p-ERK were determined and normalized against the level of si-NC cells at 60 min (n = 3 ± SD). (E&F) Cells were stimulated with EGF for indicated hours, and cell lysates were immunoblotted with the indicated antibodies. The percentages of remaining EGFR at different time points were determined. See also Figure S5.