The poly(A) polymerase Star-PAP is regulated by stably associated phosphoinositide messengers

Abstract

Star-PAP is a noncanonical poly(A) polymerase that controls gene expression. Star-PAP was previously reported to bind PIPKI⍺ and its product PI(4,5)P₂, which regulate Star-PAP activity and expression of specific genes. Recent studies have revealed a nuclear p53-phosphoinositide signaling pathway in which the phosphatidylinositol transfer proteins (PITPs) and phosphoinositide kinases/phosphatases bind p53 to sequentially modify p53-linked phosphoinositides and regulate p53 function. Here, we demonstrate that multiple phosphoinositides are also coupled to Star-PAP in response to stress. This pathway is initiated by PITP⍺/β binding to Star-PAP, and the Star-PAP-phosphoinositide complexes are sequentially modified by PI4KII⍺, PIPKI⍺, IPMK, and PTEN. The formation of Star-PAP-phosphoinositide complexes enhances the association of the small heat shock proteins HSP27 and ⍺B-crystallin with Star-PAP. Knockdown of the PITPs, PIP kinases, or HSP27 reduces the expression of Star-PAP targets. Our results demonstrate that PITP⍺/β play a key role in the assembly of Star-PAP-phosphoinositide complexes that are sequentially interconverted by PIP kinases/phosphatases and recruit the small heat shock proteins to these complexes to regulate Star-PAP activity in response to stress.

Keywords: PIP(n)-linked proteins; Star-PAP; phosphatidylinositol transfer proteins; phosphoinositide; phosphoinositide kinases; small heat shock proteins.

Figure 1

PI(4)P, PI(4,5)P₂, and PI(3,4,5)P₃ stably associated with Star-PAP under genotoxic and oxidative stress. A–C, HEK293FT cells were transiently transfected with HA-tagged Star-PAP. Forty-eight hours later, cells were treated with 0.6% DEM for 4 h and processed for IP of Star-PAP and fluorescent WB using antibodies against HA and PI(4)P (A), PI(4,5)P₂ (B), or PI(3,4,5)P₃ (C). D–F, HeLa cells treated with 100 μM etoposide or 0.6% DEM for 4 h, and PLA analysis was performed for Star-PAP and PI(4)P (C), PI(4,5)P₂ (D), or PI(3,4,5)P₃ (E). Images (100x with oil) were captured using a SP8 Leica confocal microscope (n = 3 images each from three independent experiments, Scale bar, 10 μM). PLA foci per cell in C–E were quantified using ImageJ (n = 30 cells from three independent experiments). The p values are for one-tailed two-sample t test versus vehicle. G, H, HEK293FT cells were cultured at low confluency in media containing ³H myo-inositol or unlabeled control myo-inositol. After 72 h of growth to confluency, cells were treated with 0.6% DEM for 4 h before being processed for IP against Star-PAP. Star-PAP levels were confirmed by WB (G) before samples were resolved by SDS-PAGE, and the gel lane was excised and sectioned. Gel sections were then dissolved and analyzed by liquid scintillation counting. Disintegrations per minute (DPM) were normalized to the IPed Star-PAP level (H) (n = 3 independent experiments). The p values are for one-tailed two-sample t test versus IgG control. DEM, diethyl maleate oxygenase-1; eto, etoposide; IPed, immunoprecipitated; PI(3,4,5)P_3, phosphatidylinositol 3,4,5-trisphosphate; PI(4)P, phosphatidylinositol 4-monophosphate; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; PLA, proximity ligation assay; Star-PAP, speckle-targeted PIPKIα regulated-poly(A) polymerase.

Figure 2

PITP⍺/β and PIP kinases/phosphatase associate with Star-PAP under genotoxic and oxidative stress. A–F, HeLa cells treated with 100 μM etoposide or 0.6% DEM for 4 h, and PLA analysis was performed for endogenous Star-PAP and class I PITPs PITP⍺ (A), PITPβ (B), PI4KII⍺ (C), PIPKI⍺ (D), IPMK (E), and PTEN (F). PLA foci per cell were quantified using ImageJ (n = 30 cells from three independent experiments). The p values are for one-tailed two-sample t test versus vehicle. DEM, diethyl maleate oxygenase-1; eto, etoposide; PITP, phosphatidylinositol transfer protein; PLA, proximity ligation assay; Star-PAP, speckle-targeted PIPKIα regulated-poly(A) polymerase.

Figure 3

PITP⍺/β and PIP kinases/phosphatases bind Star-PAP in response to stress. A–F, HeLa cells were treated with 100 μM etoposide or 0.6% DEM for 4 h and processed for IP of Star-PAP and WB using antibodies against PITP⍺, PITPβ, PI4KII⍺, IPMK, and PTEN (A). PITP⍺ (B), PITPβ (C), PI4KII⍺ (D), IPMK (E), and PTEN (F) protein levels were quantified and normalized to the Star-PAP level in each condition (n = 3 independent experiments). The p values are for one-tailed two-sample t test versus vehicle. G, H, in vitro binding assay was performed by adding increasing amount of His-tagged PI4KII⍺ protein to 20 nM His-tagged PITPβ protein and 500 nM His-tagged Star-PAP protein. PITPβ was then IPed and WB for PITPβ, PI4KII⍺, and Star-PAP was performed (G). Protein levels were quantified using an Odyssey Imaging System (LI-COR Biosciences, n = 3 independent experiments). The binding curve is drawn through the PI4KII⍺/PITPβ signal ratio of each condition (H). DEM, diethyl maleate oxygenase-1; eto, etoposide; IPed, immunoprecipitated; PITP, phosphatidylinositol transfer protein; Star-PAP, speckle-targeted PIPKIα regulated-poly(A) polymerase.

Figure 4

PITP⍺/β regulate PIP_n binding to Star-PAP. A, B, HeLa cells were transiently transfected with control siRNAs (siCon) or siRNAs targeting both PITP⍺/β. Forty-eight hours later, transfected cells were treated with 100 μM etoposide or 0.6% DEM for 4 h. PLA images were captured using SP8 Leica confocal microscope (100x with oil) for endogenous Star-PAP and PI(4)P (A) or PI(3,4,5)P₂ (B) (n = 3 images each from three independent experiments, Scale bar, 10 μM). PLA foci per cell were quantified using ImageJ (n = 30 cells from three independent experiments). The p values are for one-tailed two-sample t test versus control siRNA treated conditions. C, D, HEK293FT cells were transiently transfected with HA-tagged Star-PAP. Forty-eight hours later, cells were transfected with control siRNAs (siCon) or siRNAs targeting PITP⍺, PITPβ, or both PITP⍺/β. Forty-eight hours later, cells were processed for IP of Star-PAP and fluorescent WB for using antibodies against HA or PI(4,5)P₂ (C) (n = 3 independent experiments). PI(4,5)P₂ levels were quantified by Odyssey Imaging System (LI-COR Biosciences) and normalized to the HA level in each condition (D). The p values are for one-tailed two-sample t test versus control siRNA-treated condition. DEM, diethyl maleate oxygenase-1; eto, etoposide; IPed, immunoprecipitated; PI(3,4,5)P_3, phosphatidylinositol 3,4,5-trisphosphate; PI(4)P, phosphatidylinositol 4-monophosphate; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PIP_n, phosphatidylinositol phosphate; PITP, phosphatidylinositol transfer protein; PLA, proximity ligation assay; Star-PAP, speckle-targeted PIPKIα regulated-poly(A) polymerase.

Figure 5

PI4KII⍺, PIPKI⍺, IPMK, and PTEN regulate the interconversion of Star-PAP-coupled phosphoinositides. A, B, HeLa cells were transfected with control siRNAs or siRNAs targeting PI4KII⍺. Forty-eight hours later, cells were treated with 100 μM etoposide or 0.6% DEM for 4 h before performing PLA analysis of Star-PAP and PI(4)P or PI(4,5)P₂ (n = 3 images each from three independent experiments, Scale bar, 10 μM). PLA foci of Star-PAP-PI(4)P (A) and Star-PAP-PI(4,5)P₂ (B) per cell were quantified using ImageJ (n = 30 cells from three independent experiments). The p values are for one-tailed two-sample t test versus control siRNA treated conditions. C, D, HeLa cells were transfected with control siRNAs or siRNAs targeting PIPKI⍺. Forty-eight hours later, cells were treated with 100 μM etoposide or 0.6% DEM for 4 h before performing PLA analysis of Star-PAP and PI(4)P or PI(4,5)P₂ (n = 3 images each from three independent experiments, Scale bar, 10 μM). PLA foci of Star-PAP-PI(4)P (C) and Star-PAP-PI(4,5)P₂ (D) per cell were quantified using ImageJ (n = 30 cells from three independent experiments). The p values are for one-tailed two-sample t test versus control siRNA-treated conditions. E, F, MDA-MB-231 cells were transfected with control siRNAs or siRNAs targeting PIPKI⍺, IPMK, or PTEN. Forty-eight hours later, PLA analysis was performed on Star-PAP and PI(4,5)P₂ (E) or PI(3,4,5)P₃ (F) (n = 3 images each from three independent experiments, Scale bar, 10 μM). PLA foci per cell were quantified using ImageJ (n = 10 cells from three independent experiments). The p values are for one-tailed two-sample t test versus control siRNA-treated conditions. DEM, diethyl maleate oxygenase-1; eto, etoposide; PI(3,4,5)P_3, phosphatidylinositol 3,4,5-trisphosphate; PI(4)P, phosphatidylinositol 4-monophosphate; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PLA, proximity ligation assay; Star-PAP, speckle-targeted PIPKIα regulated-poly(A) polymerase.

Figure 6

Small heat shock proteins bind Star-PAP in response to stress. A, B, HeLa cells treated with 100 μM etoposide or 0.6% DEM for 4 h. Forty-eight hours later, PLA analysis was performed for Star-PAP and HSP27 (A) or ⍺B-crystallin (B) (n = 3 images each from three independent experiments, Scale bar, 10 μM). PLA foci per cell were quantified using ImageJ (n = 30 cells from three independent experiments). The p values are for one-tailed two-sample t test versus vehicle. C, HeLa cells were treated with 100 μM etoposide or 0.6% DEM for 4 h and processed for IP of Star-PAP and WB using antibodies against Star-PAP, HSP27, and ⍺B-crystallin (n = 3 independent experiments). HSP27 and ⍺B-crystallin levels were quantified by Odyssey Imaging System (LI-COR Biosciences) and normalized to the Star-PAP level in each condition. The p values are for one-tailed two-sample t test versus vehicle. D, HeLa cells were transfected with control siRNAs or siRNAs targeting PIPKI⍺. Forty-eight hours later, cells were processed for IP of Star-PAP and WB with antibodies against Star-PAP and HSP27 (n = 3 independent experiments). HSP27 levels were quantified and normalized to the Star-PAP level in each condition. The p values are for one-tailed two-sample t test versus control siRNA-treated condition. DEM, diethyl maleate oxygenase-1; eto, etoposide; PLA, proximity ligation assay; Star-PAP, speckle-targeted PIPKIα regulated-poly(A) polymerase.

Figure 7

PITP⍺/β and HSP27 regulate expression of Star-PAP targets. A, HeLa cells were transfected with control siRNAs or siRNAs targeting both PITP⍺/β or Star-PAP. Forty-eight hours later, cells were treated with 0.6% DEM for 4 h and then processed for WB using an antibody against HO-1 (n = 3 independent experiments). HO-1 levels were quantified by Odyssey Imaging System (LI-COR Biosciences) and normalized to the actin level in each condition. p-values are for one-tailed two-sample t test versus control siRNA-treated conditions. B, HEK293FT cells were transfected with control siRNAs or siRNAs targeting both PITP⍺/β or Star-PAP. Forty-eight hours later, cells were treated with 0.6% DEM for 4 h and then processed for WB with an antibody against HO-1 (n = 3 independent experiments). HO-1 levels were quantified and normalized to the actin level in each condition. The p values are for one-tailed two-sample t test versus control siRNA-treated conditions. C–F, MDA-MB-231 cells were transfected with control siRNAs or siRNAs targeting PI4KII⍺ knockdown (C), HSP27 knockdown (D), IPMK knockdown (E), or Star-PAP knockdown (F). Forty-eight hours later, cells were treated with 1 mM tBHQ or 0.6% DEM for 4 h and then processed for WB with an antibody against HO-1 (n = 3 independent experiments). HO-1 levels were quantified and normalized to the actin level in each condition. The p values are for one-tailed two-sample t test versus control siRNA-treated conditions. G–J, MDA-MB-231 cells were transfected with control siRNAs or siRNAs PI4KII⍺ knockdown (G), HSP27 knockdown (H), IPMK knockdown (I), or Star-PAP knockdown (J). Twenty-four hours later, cells were treated with 30 μM cisplatin for 24 h or 100 μM etoposide for 4 h and then processed for WB using an antibody against BIK (n = 3 independent experiments). BIK levels were quantified and normalized to the actin level in each condition. The p values are for one-tailed two-sample t test versus control siRNA-treated conditions. BIK, Bcl-2-interacting killer; DEM, diethyl maleate oxygenase-1; HO-1, heme oxygenase-1; PITP, phosphatidylinositol transfer protein; Star-PAP, speckle-targeted PIPKIα regulated-poly(A) polymerase; tBHQ, tert-butylhydroquinone.