Sprouty2 regulates PI(4,5)P2/Ca2+ signaling and HIV-1 Gag release

Abstract

We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P(2) to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P(2) and PLCγ, interfered with PI(4,5)P(2) in a manner similar to that of U73122, an inhibitor of PI(4,5)P(2) hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a crucial determinant of PI(4,5)P(2) binding in the C-terminal domain of Spry2, prevented the interference, indicating that binding to the phospholipid is required. By contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P(2) colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited wild type Gag release as virus-like particles. Disrupting either binding determinant relieved the inhibition. IP3R-mediated Ca(2+)signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P(2) and IP3R influences Spry2 function by controlling its distribution and ERK activation.

FIGURE 1. Spry2 inhibits PI(4,5)P₂ hydrolysis

Panels A–D. COS-12A2 cells were mock-treated with PBS (panel A) or incubated for 24 hr in media containing either DMSO alone (panel B) or 0.5 μM U73122 or U73343 in DMSO (panels C and D). Samples in panels B–D were prepared at 4 °C; samples in panels E–J were prepared at 23 °C. The mock-treated sample in panel A was stained for Gag with anti-capsid (CA) antibody to demonstrate that the cell line expresses Gag constitutively. Cells in panels B-J were stained with anti-PI(4,5)P₂ antibody. Panels H–J. COS-12A2 cells were transfected with DNA encoding HA-tagged Spry2. HA-Spry2 (green) and PI(4,5)P₂ (red) were visualized by indirect immunofluorescence using FITC- or TRITC-labeled secondary antibodies targeted to primary antibodies against the HA tag on Spry2 or the phospholipid, respectively. The bar measures 10 μm.

FIGURE 2. Spry2 mutants used in this study

The location of the PLCγ-binding determinant Y55 and its flanking region in the Spry2 N-terminal domain (NTD) and the PI(4,5)P₂-binding determinant R252 in the C-terminal domain (CTD) are indicated. The filled box represents the most conserved region in the otherwise highly divergent NTD.

FIGURE 3. Mapping determinants of Spry2-mediated preservation of PI(4,5)P₂

COS-12A2 cells were transfected with DNA encoding WT (panels A–C) or mutated forms of the Spry2 protein (panels D–L). Spry2 (green) and PI(4,5)P₂ (red) were visualized as described in the legend to figure 1. The bar measures 10 μm.

FIGURE 4. Co-expression of Gag with WT Spry2 inhibits VLP release; inhibition is relieved by mutation of the PI(4,5)P₂ and PLCγ-binding regions

COS-1 cells were transfected with DNA encoding WT HIV-1 Gag alone (lane 1) or with DNA encoding Gag and WT Spry2 (lanes 2 and 3), Spry2-Δ36–66 (lanes 4 and 5) or Spry2-R252D (lanes 6 and 7) at spry2-gag ratios of 1:1 or 2:1. Forty eight hours after transfection, the cells were harvested and media analyzed. Media and cell lysates were examined for VLP production (panel A) and expression of Gag-HA in the cytoplasm (panel B) by Western analysis. Panel C, Quantitative analysis of VLP release efficiency.

FIGURE 5. Depletion of the endogenous pool of IP3R-3 inhibits ERK activation

Panel A, Analysis of IP3R-3 depletion by Western analysis. Cell lysates prepared from cells transfected with DNA encoding HIV-1 Gag as described in Materials and Methods were analyzed by Western blotting for expression of IP3R-3. The blot was re-probed sequentially for p-ERK, ERK and Gag using specific antibodies directed to the proteins. Panel B, Quantitative analysis of p-ERK activation. The panel shows the ratio of the p-ERK signal in the cell lysate to the sum of the pERK plus ERK signals in the lysate.

FIGURE 6. IP3R-3 depletion alters Spry2 distribution

HeLa cells expressing Gag-GFP in cultures treated with non-targeting control siRNA (panel A) or siRNA targeted to IP3R-3 (panel B) were examined by confocal microscopy. Spry2 was detected by indirect immunofluorescence using TRITC-tagged secondary antibody. z = section through z plane of cell. Bars indicate 10 μm.

FIGURE 7. A model linking Spry2 control of Ca²⁺ release from IP3R-gated stores to IP3R control of Spry2 at the plasma membrane

The blockade to release of Ca²⁺ from the IP3R-gated stores due to the Spry2-mediated inhibition of PI(4,5)P₂ hydrolysis (red lines and font) may signal disruption of Spry2-PI(4,5)P₂ binding at the plasma membrane (green line).