A novel mechanism by which tissue transglutaminase activates signaling events that promote cell survival - PubMed (original) (raw)

A novel mechanism by which tissue transglutaminase activates signaling events that promote cell survival

Lindsey K Boroughs et al. J Biol Chem. 2014.

Abstract

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Keywords: Akt; Apoptosis; Cell Survival; PI3-kinase (PI3K); Src; Tissue Transglutaminase; Transformation; p70 S6-kinase.

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Figures

FIGURE 1.

FIGURE 1.

Ectopic expression of tTG in NIH3T3 fibroblasts promotes cell growth and survival. A, whole cell lysates of parental NIH3T3 cells, or NIH3T3 cells stably expressing the vector alone or a Myc-tagged form of tTG, were immunoblotted with tTG, Myc, and actin antibodies. The same cell lysates were also assayed for transamidation (cross-linking) activity by determining the incorporation of BPA into lysate proteins as described in “Experimental Procedures.” B, focus formation assays were carried-out on parental fibroblasts that were transiently transfected without (Mock), or with expression plasmids encoding either Myc-tagged tTG (tTG), or an HA-tagged activated form of Ras (H-Ras G12V). The cells were maintained in DMEM supplemented with 10% CS for 10 days, at which time they were fixed and stained with crystal violet. Shown are representative images of the resulting foci that formed for each condition. C, cell migration (scratch) assays were performed on NIH3T3 cells stably expressing the vector alone or a Myc-tagged form of tTG. Twenty-four hours after striking the wound, the cells were fixed and then visualized by light microscopy to determine the extent of wound closure. One set of vector alone-expressing fibroblasts was fixed immediately after striking the wound (Control 0 h.) to indicate the width of the initial wound (indicated by dashed lines). D, cultures of the NIH3T3 cells stably expressing the vector alone or a Myc-tagged form of tTG were placed in serum-free medium for the indicated lengths of time, at which point they were collected and stained with DAPI to identify condensed and/or blebbed nuclei. Percent apoptosis was determined by calculating the ratio of apoptotic to non-apoptotic cells. The experiments were performed in triplicate, and the results were averaged. The error bars indicate standard deviation, and the p values determined for the different conditions are as follows; *, p < 0.05 and **, p < 0.01. E, stable cell lines were cultured in serum free medium for the indicated lengths of time and lysed. The extracts were then immunoblotted with Myc, actin, cleaved caspase-3, and cleaved PARP antibodies. F, growth in low serum assays were performed on NIH3T3 cells stably expressing the vector alone or Myc-tagged tTG by plating them at a density of 2 × 104 cells/dish in 6-well dishes and then placing them in DMEM containing 0.1% CS. Every other day for 6 days, one set of cells was counted, while on the remaining sets of cells the medium was replenished. The experiments were performed in triplicate, and the results were averaged together and graphed. The error bars indicate standard deviation, and the p values determined for the different conditions are as follows; *, p < 0.05 and **, p < 0.01.

FIGURE 2.

FIGURE 2.

tTG promotes activation of the PI3-kinase/mTOR/p70 S6-kinase pathway. A, NIH3T3 cells stably expressing the vector alone or a Myc-tagged form of tTG were placed in serum-free medium for 24 h, at which time they were lysed and subjected to Western blot analysis using the indicated antibodies. B, serum-starved cultures of the same stable cell lines were left untreated or were treated with EGF for 10 min prior to being lysed. Immunoprecipitations using a p85 antibody (IP: p85) were carried-out on the cell extracts (1.2 mg), as was a beads only (no antibody) immunoprecipitation performed on lysates collected from EGF-stimulated NIH3T3 cells expressing the vector alone (the negative control for these experiments). The resulting immunocomplexes and 60 μg of each whole cell lysate (WCL) were subjected to Western blot analysis using the indicated antibodies (left panels). The immunocomplexes were also subjected to kinase reactions (right panel). The amount of PI(3,4,5)P3 generated by the samples was read out by ELISA. The assays were performed in triplicate, and the results were averaged together and graphed. The error bars indicate standard deviation, and the p values determined for the different conditions are as follows; *, p < 0.05. C, stable cell lines were cultured in serum-free medium supplemented without (not treated; NT) or with MDC for 24 h at which time they were lysed. The whole cell lysates were then subjected to Western blot analysis using the indicated antibodies.

FIGURE 3.

FIGURE 3.

Src activity is required for tTG-stimulated PI3-kinase activation. A, NIH3T3 cells stably expressing the vector alone or Myc-tagged tTG were placed in serum-free medium for 12 h, at which time they were treated without (not treated; NT) or with PP2 for an additional 12 h. The cells were then lysed, and the cell extracts were subjected to Western blot analysis using the indicated antibodies. B, stable cell lines were transfected without (Mock) or with an HA-tagged constitutively active form of Src (v-Src) and then were maintained in serum-free medium for 12 h. The cells were lysed, and the extracts subjected to Western blot analysis using the indicated antibodies.

FIGURE 4.

FIGURE 4.

tTG binds Src and PI3-kinase. A, immunoprecipitations were performed using an HA antibody on whole cell lysates (1.2 mg) collected from HEK293T cells ectopically expressing Myc-tagged tTG, or Myc-tagged tTG and HA-tagged c-Src. A beads only control was included to confirm the specificity of the interaction. The resulting immunocomplexes (IP:HA) and 60 μg of each whole cell lysate (WCL) were blotted with Myc and HA antibodies. B, immunoprecipitations were performed using an HA antibody on extracts (1.2 mg) collected from HEK293T cells ectopically expressing Myc-tagged tTG, or Myc-tagged tTG together with either the HA-tagged form of the p110 catalytic subunit of PI3-kinase (HA-p110) or the p85 regulatory subunit of PI3-kinase (HA-p85). A beads-only control was included to confirm the specificity of the interactions. The resulting immunocomplexes (IP:HA) and 60 μg of each whole cell lysate (WCL) were blotted with Myc and HA antibodies. C, immunoprecipitations using a Myc antibody were performed on extracts (1.2 mg) collected from NIH3T3 cells stably expressing a Myc-tagged form of tTG that were further transfected with HA-tagged forms of the p110 catalytic subunit of PI3-kinase (HA-p110), the p85 regulatory subunit of PI3-kinase (HA-p85), or c-Src (HA-c-Src). Similar immunoprecipitations were performed on extracts (1.2 mg) collected from NIH3T3 cells expressing the vector alone that were further transfected with the same constructs to confirm the specificity of the interactions. The resulting immunocomplexes (IP:Myc) and 60 μg of each whole cell lysate (WCL) were blotted with Myc and HA antibodies.

FIGURE 5.

FIGURE 5.

Inhibiting tTG or Src activity blocks the ability of tTG to bind Src and the ability of Src to phosphorylate p85. A, HEK293T cells ectopically expressing Myc-tagged tTG (Myc-tTG), Myc-tagged tTG and HA-tagged c-Src (Myc-tTG/HA-c-Src), or Myc-tagged tTG and the HA-tagged p85 regulatory subunit of PI3-kinase (Myc-tTG/HA-p85), were treated without (not treated; NT) or with PP2 or MDC for 6 h and then lysed. The cell lysates (1.2 mg) were subjected to immunoprecipitations using an HA antibody. A beads-only control was included to confirm the specificity of the interactions. The resulting immunocomplexes (IP:HA) and 60 μg of each whole cell lysate (WCL) were blotted using Myc and HA antibodies. B, immunoprecipitations were performed using an HA antibody on whole cell lysates (1.2 mg) collected from HEK293T cells ectopically expressing various combinations of Myc-tagged tTG wild-type (WT), Myc-tagged tTG C277V, and HA-tagged c-Src. A beads-only control was included to confirm the specificity of the interaction. The resulting immunocomplexes (IP:HA) and 60 μg of each whole cell lysate (WCL) were blotted with Myc and HA antibodies. C, HEK293T cells co-expressing Myc-tagged tTG and HA-tagged p85 (Myc-tTG/HA-p85) were treated without (not treated; NT) or with PP2 or MDC for 6 h and then lysed. The lysates (1.2 mg) were subjected to immunoprecipitations using an HA antibody. 60 μg of each whole cell lysate (WCL) was blotted with Myc and HA antibodies. The resulting immunocomplexes (IP:HA) were first blotted with a phosphotyrosine antibody. The blot was then stripped and re-probed with an HA antibody to confirm that an equal amount of p85 was immunoprecipitated for each condition. D, experiment shown in C was performed in triplicate and the extent of HA-tagged p85 tyrosine phosphorylation detected by Western blot analysis was quantified for each condition and averaged and graphed. The error bars represent standard deviation, and the p values determined for the different conditions are as follows; *, p < 0.05 and **, p < 0.01. E, NIH3T3 cells stably overexpressing Myc-tagged tTG were treated without (not treated; NT) or with PP2 or MDC for 12 h and then lysed. The extracts were subjected to Western blot analysis using an antibody that recognizes a tyrosine-phosphorylated form of p85 (p-p85 (Y458)), as well as ones that detect total p85 (pan-p85), Myc-tTG, and actin.

FIGURE 6.

FIGURE 6.

Inhibition of Src, PI3-kinase, or mTORC1 blocks the tTG-enhanced cell survival. A and B, NIH3T3 cells stably expressing the vector alone or Myc-tagged tTG were placed in serum-free medium supplemented without (not treated; NT) or with PP2, LY294002, MDC, or rapamycin for 36 h, at which time the cells were collected and stained with DAPI to identify condensed and/or blebbed nuclei. Percent apoptosis was determined by calculating the ratio of apoptotic to non-apoptotic cells. The experiments were performed in triplicate, and the results were averaged. The error bars indicate standard deviation, and the p values determined for the different conditions are as follows; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

FIGURE 7.

Model depicting how tTG promotes c-Src-mediated PI3-kinase signaling and cell survival. Our results suggest that tTG functions to promote cell survival by potentiating the activation of PI3-kinase. Specifically, tTG forms a complex with c-Src and PI3-kinase, an outcome that promotes a c-Src-dependent phosphorylation of the p85 regulatory subunit of PI3-kinase causing it to adopt a conformation that allows the p110 catalytic subunit of PI3-kinase to become activated and signal to its downstream effectors.

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