Nuclear translocation controlled by alternatively spliced isoforms inactivates the QUAKING apoptotic inducer

Abstract

The quaking viable mice have myelination defects and develop a characteristic tremor 10 d after birth. The quaking gene encodes at least five alternatively spliced QUAKING (QKI) isoforms that differ in their C-terminal 8--30-amino-acid sequence. The reason for the existence of the different QKI isoforms and their function are unknown. Here we show that only one QKI isoform, QKI-7, can induce apoptosis in fibroblasts and primary rat oligodendrocytes. Heterodimerization of the QKI isoforms results in the nuclear translocation of QKI-7 and the suppression of apoptosis. The unique C-terminal 14 amino acids of QKI-7 confers the ability to induce apoptosis to heterologous proteins such as the green fluorescent protein and a QKI-related protein, Caenorhabditis elegans GLD-1. Thus, the unique C-terminal sequences of QKI-7 may function as a life-or-death 'sensor' that monitors the balance between the alternatively spliced QKI isoforms. Moreover, our findings suggest that nuclear translocation is a novel mechanism of inactivating apoptotic inducers.

Figure 1

QKI-7 is a potent apoptotic inducer. (A) Detection of early apoptosis by annexin V. Myc–QKI-5 or myc–QKI-7 were transfected into NIH 3T3 cells and stained with annexin V–FITC and propidium iodide (PI). The cells were visualized by fluorescence microscopy. The arrowheads align the cells in top and bottom panels. (B) Myc–QKI-5 or myc–QKI-7 were transfected into NIH 3T3 cells stained live with annexin V–FITC and fixed, and the myc-tagged QKI-7 was visualized by indirect immunofluorescence with anti-myc antibody followed by a rhodamine-conjugated secondary antibody. (C) Suppression of apoptosis by Bcl-2 overexpression. GFP alone or GFP–QKI-7 were cotransfected into NIH 3T3 cells with increasing amounts of a Bcl-2 expression vector (pCEP4–Bcl-2) or empty plasmid (pCEP4). After 12 h (solid bars) and 36 h (open bars) the cells were fixed and stained with DAPI to visualize the apoptotic nuclei. (D) The caspase inhibitor Z-DEVD.fmk suppresses QKI-7-induced apoptosis. GFP or GFP–QKI-7 were transfected into NIH 3T3 cells and treated with either DMSO (control) or 50 μM Z-DEVD.fmk as indicated. After 12 h (solid bars) and 36 h (open bars) the cells were fixed and stained with DAPI to visualize the apoptotic nuclei. (C,D) The presence of apoptotic nuclei was scored as cells undergoing apoptosis and expressed as % green cells. Each bar represents the mean + S.E. of 3 experiments (n > 450, n = number of cells counted). Statistical evaluation was calculated by paired Student's t-test. (*) Values that differ significantly from GFP at 36 h (P < 0.01).

Figure 2

The C-terminal 14 amino acids of QKI-7 induce apoptosis. (A) A schematic diagram of GFP fusion proteins is shown at left. The GSG, KH, NK, and CK regions are indicated. The striped box at the C terminus denotes the unique sequences of the QKI alternatively spliced isoforms. NLS represents the SV40 large T antigen nuclear localization signal. Expression plasmids encoding these GFP fusion proteins were transfected in NIH 3T3 cells. After 12 h (solid bars) and 36 h (open bars) the cells were fixed and stained with DAPI to visualize the apoptotic nuclei and expressed as a percentage of green cells. The localization of each protein at 12 h is indicated at right. (C) A predominant cytoplasmic localization; (N) a predominant nuclear localization. Each bar represents the mean + S.E. of 3 experiments (n > 450). (*) Values that differ significantly from GFP at 36 h (*, P < 0.01). (B) The amino acids of the unique sequences of the different QKI isoforms used. (C) C6 glioma cells were transfected with the indicated amount of the expression plasmids encoding GFP–QKI-7 or GFP:14. After 12 and 36 h the cells were fixed and stained with DAPI to visualize the apoptotic nuclei and expressed as a percentage of green cells. Each bar represents the mean + S.E. of three experiments (n > 300).

Figure 3

Expression of the QKI isoforms. (A) Untransfected (control) or myc–QKI-5, myc–QKI-6, myc–QKI-7, and myc–QKI-G were expressed in HeLa cells. The cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-myc (lanes 1–5) or anti-pan QKI antibodies (lanes 6–10). The molecular mass markers are shown at left in kD. (B) HeLa cells were transfected with the indicated myc–QKI as in A and analyzed by immunoblotting with anti-QKI-5 (lanes 1–4), anti-QKI-6 (lanes 5–8), and anti-QKI-7 (lanes 9–12) antibodies. The migration of QKI-5, QKI-6, and QKI-7 is indicated. (C) NIH 3T3 cells were transfected with expression plasmids expressing the indicated GFP fusion protein. The cells were lysed and analyzed by immunoblotting with anti-pan QKI antibodies.

Figure 3

Expression of the QKI isoforms. (A) Untransfected (control) or myc–QKI-5, myc–QKI-6, myc–QKI-7, and myc–QKI-G were expressed in HeLa cells. The cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-myc (lanes 1–5) or anti-pan QKI antibodies (lanes 6–10). The molecular mass markers are shown at left in kD. (B) HeLa cells were transfected with the indicated myc–QKI as in A and analyzed by immunoblotting with anti-QKI-5 (lanes 1–4), anti-QKI-6 (lanes 5–8), and anti-QKI-7 (lanes 9–12) antibodies. The migration of QKI-5, QKI-6, and QKI-7 is indicated. (C) NIH 3T3 cells were transfected with expression plasmids expressing the indicated GFP fusion protein. The cells were lysed and analyzed by immunoblotting with anti-pan QKI antibodies.

Figure 3

Expression of the QKI isoforms. (A) Untransfected (control) or myc–QKI-5, myc–QKI-6, myc–QKI-7, and myc–QKI-G were expressed in HeLa cells. The cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-myc (lanes 1–5) or anti-pan QKI antibodies (lanes 6–10). The molecular mass markers are shown at left in kD. (B) HeLa cells were transfected with the indicated myc–QKI as in A and analyzed by immunoblotting with anti-QKI-5 (lanes 1–4), anti-QKI-6 (lanes 5–8), and anti-QKI-7 (lanes 9–12) antibodies. The migration of QKI-5, QKI-6, and QKI-7 is indicated. (C) NIH 3T3 cells were transfected with expression plasmids expressing the indicated GFP fusion protein. The cells were lysed and analyzed by immunoblotting with anti-pan QKI antibodies.

Figure 4

The localization of the transfected QKI isoforms in HeLa cells. Expression plasmids expressing GFP (A), GFP–QKI-5 (B), GFP–QKI-6 (C), GFP–QKI-7 (D), or GFP–QKI-G (E) were transfected in HeLa cells. After 12 h the cells were fixed and visualized using fluorescence microscopy.

Figure 5

Characterization of the apoptosis induced by the QKI isoforms. (A) QKI-7 is the only QKI apoptotic inducer. NIH 3T3 cells were transfected with expression vectors encoding the indicated GFP fusion protein. After 12 h (solid bars) and 36 h (open bars) the cells were fixed and stained with DAPI. The % green cells represents the number of transfected cells as a percentage of the total number of cells as visualized by DAPI. Each bar represents the mean + S.E. of 3 experiments (n > 450). (*) Values that differ significantly from GFP at 36 h (P < 0.01). (B) Dimerization is required for QKI-5 or QKI-6 to suppress the apoptosis induced by QKI-7. NIH 3T3 cells were cotransfected with expression plasmids encoding the GFP proteins indicated and the data were expressed as in A.

Figure 6

QKI-7-induced apoptosis is suppressed by QKI-5. (A) NIH 3T3 cells were transfected with an expression plasmid that encodes myc–QKI-7 alone (A,B) or with expression vectors for GFP (C–E), GFP–QKI:1–311 (F–H), or GFP–QKI-5 (I–K). After 36 h the cells were fixed and visualized by indirect immunofluorescence by using the anti-myc antibody followed by rhodamine-conjugated secondary antibody. DAPI was used to visualize the nuclei. Each column represents the same field of cells as visualized under the green (top), red (middle), and blue (bottom) filters. The arrows are used to align the panels. (B) Quantitation of the apoptosis suppressed by QKI-5. NIH 3T3 cells were transfected as in A. For the cells expressing both a GFP fusion protein and a myc epitope-tagged protein, the apoptotic cells were expressed as a percentage of the cells that were both green and red. The panel at right indicates the percentage of cells that were transfected from the total number of cells. Each bar represents the mean + S.E. of 3 experiments (n > 450). (*) Values that differ significantly from GFP at 36 h (P < 0.01).

Figure 7

Heterodimerization of the QKI isoforms in fibroblasts is abolished by the E→G amino acid substitution. (A,C) HeLa cells were transfected with myc–QKI and HA–QKI isoforms as indicated. The cells were lysed and immunoprecipitated with IgG as a control (C) or anti-myc antibodies (myc). The bound proteins as well as an aliquot of total cellular lysate (TCL) were separated by SDS-PAGE and analyzed by immunoblotting with anti-HA antibodies. The migration of the heavy chain of IgG and the HA–QKI isoforms is shown. (B,D) The total cellular lysate of panels A and C were confirmed for the equivalent expression of the myc epitope-tagged QKI proteins.

Figure 8

Nuclear translocation of QKI-7 with QKI-5 or QKI-6 but not with QKI-5:E→G or QKI-6:E→G. Myc–QKI-7 was either transfected alone (A,B) or cotransfected with GFP–QKI-5 (C–E), GFP–QKI-6 (F–H), GFP–QKI-5:E→G (I–K), or GFP–QKI-6:E→G (L–N) into HeLa cells. After 12 h the cells were fixed, immunostained with an anti-myc antibody followed by a rhodamine-conjugated secondary antibody, and mounted onto a glass slide in the presence of the nuclear stain DAPI. The cells were visualized by fluorescence microscopy. Each column represent the same field of cells as visualized under the green (top), red (middle), and blue (bottom) filters. The arrows are used to align the panels.

Figure 9

Adenoviruses expressing QKI-7 induce apoptosis in primary rat OLs. (A) The generation of QKI-inducible adenoviruses. HeLa cells were coinfected at an M.O.I. of 10 with an adenovirus that constitutively expresses the tTA and a tetracycline-inducible adenovirus expressing myc–QKI-5, myc–QKI-6, or myc–-QKI-7. (+) Absence of doxycyclin; (−) addition of 1 μg/mL of doxycyclin. Essentially 100% of the cells were green 36 h after infection (not shown). The cells were harvested, lysed in sample buffer, separated by SDS-PAGE, and immunoblotted with anti-myc antibodies. pAdTR5 denotes an adenovirus generated with vector alone. pAdTR5–QKI-5, pAdTR5–QKI-6, and pAdTR5–QKI-7 represent the adenoviruses expressing QKI-5, QKI-6, and QKI-7, respectively. (B) Phase contrast photograph of a typical primary rat OLs preparation 2 d after maturation in growth factors (left panel). Uninfected OLs were stained live with anti-galactocerebroside FITC-conjugated antibodies and analyzed by flow cytometry. (C) Primary rat OLs were coinfected as in A, fixed, permeabilized, and stained with anti-myc antibodies followed by a rhodamine-conjugated secondary antibody. The cells were visualized by fluorescence microscopy. (D) Primary rat OLs were coinfected as in A, stained live with annexin V–phycoerythrin, and analyzed by flow cytometry. The percentage of GFP and annexin V positive cells is indicated for each condition. The experiment shown is representative of three experiments.