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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.


Related in: MedlinePlus

Protein stability of HA-tagged PinX1 derivatives in HeLa cells. (A) Protein stability of PinX1 truncations. HeLa cells transfected with PinX1 truncations for 48 h were treated with cycloheximide for the indicated times, followed by immunoblot. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin level was quantified as the ratio relative to the level at the 0 h time point, and the error bars were derived from three independent experiments. (C) Protein stability of PinX1 truncations with a shorter exposure time to CHX. Cells were prepared as described in A and treated with cycloheximide for the indicated times, followed by immunoblot. GAPDH was used as a loading control. (D). Quantification of PinX1 protein represented in C. PinX1 level normalized to GAPDH level was quantified as described in B. Error bars were derived from two independent experiments.
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f2-molce-38-9-814: Protein stability of HA-tagged PinX1 derivatives in HeLa cells. (A) Protein stability of PinX1 truncations. HeLa cells transfected with PinX1 truncations for 48 h were treated with cycloheximide for the indicated times, followed by immunoblot. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin level was quantified as the ratio relative to the level at the 0 h time point, and the error bars were derived from three independent experiments. (C) Protein stability of PinX1 truncations with a shorter exposure time to CHX. Cells were prepared as described in A and treated with cycloheximide for the indicated times, followed by immunoblot. GAPDH was used as a loading control. (D). Quantification of PinX1 protein represented in C. PinX1 level normalized to GAPDH level was quantified as described in B. Error bars were derived from two independent experiments.

Mentions: To address the possibility that PinX1 truncation mutants have different levels of protein stability, stability assay was performed as follows: HeLa cells were transfected with HA-PinX11–328, HA-PinX11–204, or HA-PinX1205–328, and then treated with cycloheximide (CHX) after 48 h for the indicated times to block protein biosynthesis. The level of HA-tagged PinX1 protein was monitored by immunoblot (Figs. 2A and 2B). HA-PinX1205–328 remained abundant upon increased exposure time to CHX, whereas HA-PinX11–204 was rapidly degraded. The assay performed with a shorter time of exposure to CHX confirmed different levels of stability according to the truncated form of PinX1 (Figs. 2C and 2D). HA-PinX11–328 was degraded relatively faster than HA-PinX1205–328, but slower than HA-PinX11–204 (Fig. 2). These results led us to speculate that the N-terminal region of PinX1 may negatively affect the stability of the protein. To test this possibility, GFP-fused or myc-tagged PinX11–328 was subjected to stability assays (Supplementary Fig. S1). Unlike HA- PinX1, both GFP-PinX1 and myc-PinX1 showed almost equal stability to the C-terminal of PinX1, suggesting that the HA-tag rather than the N-terminal of PinX1 exerted a negative effect on the stability of full-length PinX1. We do not fully understand how the HA tag could reduce the stability of the full length of PinX1. Nevertheless, consistent with the findings of HA-tagged PinX1 truncations, myc-PinX1205–328 was more stable than myc-PinX11–204 (Supplementary Fig. S1).


Increased Stability of Nucleolar PinX1 in the Presence of TERT.

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

Protein stability of HA-tagged PinX1 derivatives in HeLa cells. (A) Protein stability of PinX1 truncations. HeLa cells transfected with PinX1 truncations for 48 h were treated with cycloheximide for the indicated times, followed by immunoblot. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin level was quantified as the ratio relative to the level at the 0 h time point, and the error bars were derived from three independent experiments. (C) Protein stability of PinX1 truncations with a shorter exposure time to CHX. Cells were prepared as described in A and treated with cycloheximide for the indicated times, followed by immunoblot. GAPDH was used as a loading control. (D). Quantification of PinX1 protein represented in C. PinX1 level normalized to GAPDH level was quantified as described in B. Error bars were derived from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588725&req=5

f2-molce-38-9-814: Protein stability of HA-tagged PinX1 derivatives in HeLa cells. (A) Protein stability of PinX1 truncations. HeLa cells transfected with PinX1 truncations for 48 h were treated with cycloheximide for the indicated times, followed by immunoblot. (B) Quantification of PinX1 protein represented in A. PinX1 level normalized to β-actin level was quantified as the ratio relative to the level at the 0 h time point, and the error bars were derived from three independent experiments. (C) Protein stability of PinX1 truncations with a shorter exposure time to CHX. Cells were prepared as described in A and treated with cycloheximide for the indicated times, followed by immunoblot. GAPDH was used as a loading control. (D). Quantification of PinX1 protein represented in C. PinX1 level normalized to GAPDH level was quantified as described in B. Error bars were derived from two independent experiments.
Mentions: To address the possibility that PinX1 truncation mutants have different levels of protein stability, stability assay was performed as follows: HeLa cells were transfected with HA-PinX11–328, HA-PinX11–204, or HA-PinX1205–328, and then treated with cycloheximide (CHX) after 48 h for the indicated times to block protein biosynthesis. The level of HA-tagged PinX1 protein was monitored by immunoblot (Figs. 2A and 2B). HA-PinX1205–328 remained abundant upon increased exposure time to CHX, whereas HA-PinX11–204 was rapidly degraded. The assay performed with a shorter time of exposure to CHX confirmed different levels of stability according to the truncated form of PinX1 (Figs. 2C and 2D). HA-PinX11–328 was degraded relatively faster than HA-PinX1205–328, but slower than HA-PinX11–204 (Fig. 2). These results led us to speculate that the N-terminal region of PinX1 may negatively affect the stability of the protein. To test this possibility, GFP-fused or myc-tagged PinX11–328 was subjected to stability assays (Supplementary Fig. S1). Unlike HA- PinX1, both GFP-PinX1 and myc-PinX1 showed almost equal stability to the C-terminal of PinX1, suggesting that the HA-tag rather than the N-terminal of PinX1 exerted a negative effect on the stability of full-length PinX1. We do not fully understand how the HA tag could reduce the stability of the full length of PinX1. Nevertheless, consistent with the findings of HA-tagged PinX1 truncations, myc-PinX1205–328 was more stable than myc-PinX11–204 (Supplementary Fig. S1).

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.


Related in: MedlinePlus