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Increased Stability of Nucleolar PinX1 in the Presence of TERT.

Keo P, Choi JS, Bae J, Shim YH, Oh BK - Mol. Cells (2015)

Bottom Line: Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells.However, PinX1(1-204) was degraded regardless of the TERT status.These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea.

ABSTRACT
PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1(205-328), a nucleolar fragment, was much more stable than the N-terminal of PinX1(1-204), a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX1(1-328) and PinX1(205-328), nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX1(1-204) was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

No MeSH data available.


TERT stabilizes the nucleolar form of PinX1. (A) Depletion of TERT reduces the stability of nucleolar forms of PinX1. HeLa cells were transfected with siTERT, and 24 h later, cells were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with cycloheximide (CHX) for the indicated times. PinX1 truncations were detected with anti-HA antibody, and GAPDH was used as an internal control. (B) Quantification of PinX1 represented in A. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. (C) qRT–PCR confirming TERT status in the samples used in A. qRT–PCR was performed with SyBR Green reagents, and β-actin was used as a control. (D) Excess TERT stabilizes nucleolar forms of PinX1. HeLa cells transfected with TERT-myc for 24 h were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with CHX for the indicated times, and lysates were prepared for immnoblot. TERT-myc was indicated by asterisks. (E) Quantification of PinX1 protein represented in D. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. Error bars shown in graphs B, C, and E represent the S.D. of the mean from three independent experiments.
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f5-molce-38-9-814: TERT stabilizes the nucleolar form of PinX1. (A) Depletion of TERT reduces the stability of nucleolar forms of PinX1. HeLa cells were transfected with siTERT, and 24 h later, cells were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with cycloheximide (CHX) for the indicated times. PinX1 truncations were detected with anti-HA antibody, and GAPDH was used as an internal control. (B) Quantification of PinX1 represented in A. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. (C) qRT–PCR confirming TERT status in the samples used in A. qRT–PCR was performed with SyBR Green reagents, and β-actin was used as a control. (D) Excess TERT stabilizes nucleolar forms of PinX1. HeLa cells transfected with TERT-myc for 24 h were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with CHX for the indicated times, and lysates were prepared for immnoblot. TERT-myc was indicated by asterisks. (E) Quantification of PinX1 protein represented in D. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. Error bars shown in graphs B, C, and E represent the S.D. of the mean from three independent experiments.

Mentions: Next, it was examined whether the TERT-mediated increase in PinX1 stability varies according to the PinX1 truncations by measuring the stability of the PinX1 truncation mutants in TERT-depleted and -overexpressing cells (Fig. 5). HeLa cells were co-transfected with the PinX1 truncations along with si-TERT or TERT-myc, and then treated with CHX for the indicated times. Like endogenous PinX1, HA-PinX11–328 and HA-PinX1205–328 were degraded more rapidly in the TERT-depleted cells (Figs. 5A and 5B) and were more stable in the TERT-myc-expressing cells (Figs. 5D and 5E) compared to each of the control cells. However, HA-PinX11–204 was degraded in both the TERT-depleted and -overexpressing cells, with no difference to the control cells (Fig. 5). TERT status in the samples was verified by either qRT-PCR (Fig. 5C) or immunoblot (Fig. 5D), and expression of TERT-myc in the reaction of PinX1205–328 (Fig. 5D) was also verified by qRT-PCR (data not shown). Collectively, our findings indicate that TERT stabilizes the nucleolar form of PinX1, not the nuclear form.


Increased Stability of Nucleolar PinX1 in the Presence of TERT.

Keo P, Choi JS, Bae J, Shim YH, Oh BK - Mol. Cells (2015)

TERT stabilizes the nucleolar form of PinX1. (A) Depletion of TERT reduces the stability of nucleolar forms of PinX1. HeLa cells were transfected with siTERT, and 24 h later, cells were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with cycloheximide (CHX) for the indicated times. PinX1 truncations were detected with anti-HA antibody, and GAPDH was used as an internal control. (B) Quantification of PinX1 represented in A. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. (C) qRT–PCR confirming TERT status in the samples used in A. qRT–PCR was performed with SyBR Green reagents, and β-actin was used as a control. (D) Excess TERT stabilizes nucleolar forms of PinX1. HeLa cells transfected with TERT-myc for 24 h were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with CHX for the indicated times, and lysates were prepared for immnoblot. TERT-myc was indicated by asterisks. (E) Quantification of PinX1 protein represented in D. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. Error bars shown in graphs B, C, and E represent the S.D. of the mean from three independent experiments.
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f5-molce-38-9-814: TERT stabilizes the nucleolar form of PinX1. (A) Depletion of TERT reduces the stability of nucleolar forms of PinX1. HeLa cells were transfected with siTERT, and 24 h later, cells were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with cycloheximide (CHX) for the indicated times. PinX1 truncations were detected with anti-HA antibody, and GAPDH was used as an internal control. (B) Quantification of PinX1 represented in A. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. (C) qRT–PCR confirming TERT status in the samples used in A. qRT–PCR was performed with SyBR Green reagents, and β-actin was used as a control. (D) Excess TERT stabilizes nucleolar forms of PinX1. HeLa cells transfected with TERT-myc for 24 h were transfected with HA-tagged PinX1 truncations for another 24 h, and then treated with CHX for the indicated times, and lysates were prepared for immnoblot. TERT-myc was indicated by asterisks. (E) Quantification of PinX1 protein represented in D. PinX1 level normalized to GAPDH was quantified relative to the level at 0 h. Error bars shown in graphs B, C, and E represent the S.D. of the mean from three independent experiments.
Mentions: Next, it was examined whether the TERT-mediated increase in PinX1 stability varies according to the PinX1 truncations by measuring the stability of the PinX1 truncation mutants in TERT-depleted and -overexpressing cells (Fig. 5). HeLa cells were co-transfected with the PinX1 truncations along with si-TERT or TERT-myc, and then treated with CHX for the indicated times. Like endogenous PinX1, HA-PinX11–328 and HA-PinX1205–328 were degraded more rapidly in the TERT-depleted cells (Figs. 5A and 5B) and were more stable in the TERT-myc-expressing cells (Figs. 5D and 5E) compared to each of the control cells. However, HA-PinX11–204 was degraded in both the TERT-depleted and -overexpressing cells, with no difference to the control cells (Fig. 5). TERT status in the samples was verified by either qRT-PCR (Fig. 5C) or immunoblot (Fig. 5D), and expression of TERT-myc in the reaction of PinX1205–328 (Fig. 5D) was also verified by qRT-PCR (data not shown). Collectively, our findings indicate that TERT stabilizes the nucleolar form of PinX1, not the nuclear form.

Bottom Line: Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells.However, PinX1(1-204) was degraded regardless of the TERT status.These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea.

ABSTRACT
PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1(205-328), a nucleolar fragment, was much more stable than the N-terminal of PinX1(1-204), a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX1(1-328) and PinX1(205-328), nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX1(1-204) was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

No MeSH data available.