Pericentrosomal localization of the TIG3 tumor suppressor requires an N-terminal hydrophilic region motif

Abstract

Tazarotene-induced gene 3 (TIG3) is a tumor suppressor protein that has a key role in controlling cell proliferation. TIG3 is observed at reduced levels in epidermal squamous cell carcinoma, and the restoration of expression in skin cancer cells reduces cell survival. TIG3 suppresses cell survival through mechanisms that involve localization at the plasma membrane and at the centrosome. TIG3 interacts at the plasma membrane to activate enzymes involved in keratinocyte terminal differentiation, and at the centrosome to inhibit daughter centrosome separation during mitosis leading to cessation of cell proliferation and induction of apoptosis. An important goal is identifying the motifs required for TIG3 localization at these intracellular sites as a method to understand the function of TIG3 at each location. TIG3 encodes an N-terminal hydrophilic region (amino acids 1-135) and a C-terminal membrane-anchoring domain (amino acids 135-164). We show that the C-terminal hydrophobic domain targets intact TIG3 to the plasma membrane, but when isolated as an independent element localizes at the mitochondria. We further demonstrate that a segment of the N-terminal hydrophilic region targets the centrosome. These studies provide important insights regarding the mechanisms that guide subcellular localization of this keratinocyte survival regulator.

Fig. 1

TIG3 C-terminal domain is required for appropriate intracellular localization and function. A TIG3 protein domain structure. Purple indicates the N-terminal hydrophilic region and green the C-terminal hydrophobic region. Regions of the protein that are conserved in other H-rev107 family members are shown in orange. The N-terminal Homology Domain, and the NCEHFV and LRYG motifs are shown (Deucher et al., 2000). B Subcellular localization of TIG3_1–164 and TIG_1–134. SCC-13 cells were infected with 10 MOI TIG3_1–164 or 50 MOI TIG3_1–134 adenovirus and after 24 h the cells were fixed and stained with anti-TIG3 and appropriate secondary antibody (green). The sections were also stained with anti-pericentrin and appropriate secondary (red). The yellow signal indicates co-localization of TIG3 and pericentrin, red indicates absence of co-localization. Arrows indicate the centrosomes. Bars = 10 µm. C Impact of full-length TIG3_1–164 and TIG3_1–134 on cell survival. Cells were infected with 10 MOI of empty (EV) and TIG3_1–164 and TIG3_1–134 encoding adenoviruses and at 24 h post-infection the cells were harvested for cell count. The open bar indicates cell number at the time of virus delivery (t = 0) and the shaded bars indicated cell number at 24 h later. The values are mean ± SEM. Single asterisks indicate a significant increase in cell number (n = 3, p < 0.05) as compared to the control, while the double asterisks indicate reduced cell number compared to the other 24 h data points (n = 3, p < 0.05) as determined using the Student’s t-test. D Impact of TIG3_1–164 and TIG3_1–134 on SCC-13 cell apoptosis. SCC-13 cells were infected with the indicated adenovirus and after 24 h the cells were harvested for assay of caspase and PARP status. The arrows indicate TIG3 monomers and the bracket indicated high molecular weight crosslinked TIG3 as previously described (Eckert et al., 2009; Jans et al., 2008; Sturniolo et al., 2005; Sturniolo et al., 2003).

Fig. 2

Role of the C- and N-terminal domains in guiding subcellular localization. A Schematic of TIG3-EGFPN1 fusion proteins. The proteins are as described in Fig. 1A, and the blue rectangle designates EGFPN1. B Subcellular localization of TIG3_1–134-EGFPN1 and TIG3_134–164-EGFPN1 in SCC-13 cells. SCC-13 cells were electroporated with 3 µg of the indicated plasmid and after 24 h EGFPN1 fluorescence was detected by confocal microscopy. The arrows indicate the novel subcellular distribution of TIG3_134–164-EGPN1. No signal was observed in non-electroporated cells (not shown). C TIG3_134–164-EGFPN1 localizes at the mitochondria. Cells were electroporated with 3 µg of TIG3_134–164-EGFPN1 and after 24 h the cells were fixed and stained with anti-pericentrin (centrosome), GM130 (Golgi), or calnexin (ER), or incubated with MitoTracker (mitochondria) (red). The images were then visualized by confocal microscopy. The merged images are the composite of the green and red channels and yellow indicates co-localization. Arrows indicate perinuclear TIG3 localization. Red, green (EGFPN1) and merged channels are shown. Bars = 10 µm.

Fig. 3

Presence of putative centrosome localization signal in the TIG3 N-terminal hydrophilic region. A Schematic showing the TIG3 fusion proteins in which segments of the TIG3 N-terminal hydrophilic region are fused to EGFPN1 (blue). The TIG3 domains are as described in Fig. 1A. B Subcellular distribution of fusion proteins. SCC-13 cells were electroporated with 3 µg of each indicated plasmid and after 24 h the EGFPN1 signal was detected by confocal microscopy (green) and pericentrin was detected with anti-pericentrin and appropriate secondary antibody (red). Co-localization of TIG3 mutant and pericentrin is indicated by the yellow signal (arrows). TIG3_75–134, TIG3_84–134, TIG3_93–134, TIG3_102–134, and TIG3_102–125 co-localize with pericentrin at a pericentrosomal location (arrows). Bars = 10 µm. C Expression level of TIG3-EGFPN1 fusion proteins. SCC-13 cells were transfected with 3 µg of each construct and after 24 h the cells were harvested and cell extract was prepared and incubated with anti-GFP and appropriate secondary antibody (left panel). As a comparison SCC-13 cells were infected with 10 MOI of tAd5-TIG3 adenovirus and after 24 h extracts were prepared for electrophoresis and detection with anti-TIG3 (right panel). An arrow indicates the TIG3 monomer and the brackets indicate crosslinked forms of TIG3 (Eckert et al., 2009; Jans et al., 2008; Sturniolo et al., 2005; Sturniolo et al., 2003).

Fig. 4

Subcellular localization of TIG3-FLAG fusion proteins. A Schematic of the fusion proteins in which the segments of the N-terminal hydrophilic region are fused to FLAG epitope. The TIG3 domains are described in the legend to Fig. 1A. B SCC-13 cells were electroporated with 3 µg of each TIG3-FLAG fusion protein and after 24 h the cells were fixed and co-stained with the centrosome marker, pericentrin (green), and Cy3-conjugated anti-FLAG (red). Each of these fusion proteins localize at the centrosome (yellow, arrows). Bars = 10 µm. C TIG3(102–125)-FLAG accumulates at the centrosome in A431 cells and normal foreskin keratinocytes (KERn). Cells were electroporated with plasmid encoding TIG3(102–125)-FLAG and then stained with antibodies as indicated above. Bars = 10 µM.

Fig. 5

Detailed analysis of the centrosome localization motif. A Role of highly-conserved amino acids in the centrosome-localization motif. pTIG3_75–134-EGFPN1 encodes amino acids 75–134 of TIG3 fused to the N-terminus of EGFPN1. This plasmid was used as a substrate to generate plasmids encoding the indicated mutated sequences. Conserved sequences are indicated in red and mutated sequences are underlined in red. The distribution of TIG3_102–125-EGFPN1 is shown in Fig. 3B. B SCC-13 cells were electroporated with 3 µg of each plasmid and after 24 h the cells were fixed and imaged by confocal microscopy. One of the mutants, N112F/C113G, localizes with punctate structures (arrow), while the others distribute throughout the cytosol. TIG3 mutant staining (anti-FLAG) is green and anti-pericentrin staining is red. Bars = 10 µm. C The N112F/C113G mutant distributes to the mitochondria. SCC-13 cells, expressing the N112F/C113G mutant (green), were incubated with MitoTracker dye to stain mitochondria (red) and imaged by confocal microscopy. The arrows show localization of the N112F/C113G mutant and the yellow color in the merged image indicates co-localization of this mutant with MitoTracker. Bars = 10 µm. D Expression of the mutant proteins. Cells were transfected with 3 µg of each plasmid and after 24 h lysates were prepared for immunoblot detection of each mutant using anti-GFP antibody. Similar results were observed in each of three experiments.