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HILI destabilizes microtubules by suppressing phosphorylation and Gigaxonin-mediated degradation of TBCB

View Article: PubMed Central - PubMed

ABSTRACT

Human PIWIL2, aka HILI, is a member of PIWI protein family and overexpresses in various tumors. However, the underlying mechanisms of HILI in tumorigenesis remain largely unknown. TBCB has a critical role in regulating microtubule dynamics and is overexpressed in many cancers. Here we report that HILI inhibits Gigaxonin-mediated TBCB ubiquitination and degradation by interacting with TBCB, promoting the binding between HSP90 and TBCB, and suppressing the interaction between Gigaxonin and TBCB. Meanwhile, HILI can also reduce phosphorylation level of TBCB induced by PAK1. Our results showed that HILI suppresses microtubule polymerization and promotes cell proliferation, migration and invasion via TBCB for the first time, revealing a novel mechanism for HILI in tumorigenesis.

No MeSH data available.


HILI suppresses microtubule polymerization in a TBCB-dependent manner.(A) HILI down-regulated acetylation level of α-tubulin and had no significant effect on α-tubulin expression at protein level in HeLa and HepG2 cells. HeLa and HepG2 cells were transfected with MYC-HILI, sh-NC, or sh-HILI vector. After 48 h, cell lysates were prepared for Western blotting with AC-α-tubulin and α-tubulin antibody (AC, AC-α-tubulin). (B) Immunofluorescent staining of AC-α-tubulin and α-tubulin in transfected cells. (C) In vivo tubulin polymerization assays in HeLa and HepG2 cells. Supernatant (S) and pellet (P) fractions of cell lysates were analyzed with anti-α-tubulin, data were presented as mean ± sd. (*P < 0.05). (D) Knockdown of TBCB recovered acetylation level of α-tubulin decreased by HILI overexpression, overexpression of TBCB inhibited the increase of acetylation level of α-tubulin induced by HILI knockdown. (E) Immunofluorescence assays showed that HILI down-regulated acetylation level of α-tubulin in a TBCB-dependent manner (a, NC; b, HILI; c, sh-TBCB; d, HILI/sh-TBCB; e, sh-HILI; f, TBCB; g, sh-HILI/TBCB; AC, AC-α-tubulin).
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f1: HILI suppresses microtubule polymerization in a TBCB-dependent manner.(A) HILI down-regulated acetylation level of α-tubulin and had no significant effect on α-tubulin expression at protein level in HeLa and HepG2 cells. HeLa and HepG2 cells were transfected with MYC-HILI, sh-NC, or sh-HILI vector. After 48 h, cell lysates were prepared for Western blotting with AC-α-tubulin and α-tubulin antibody (AC, AC-α-tubulin). (B) Immunofluorescent staining of AC-α-tubulin and α-tubulin in transfected cells. (C) In vivo tubulin polymerization assays in HeLa and HepG2 cells. Supernatant (S) and pellet (P) fractions of cell lysates were analyzed with anti-α-tubulin, data were presented as mean ± sd. (*P < 0.05). (D) Knockdown of TBCB recovered acetylation level of α-tubulin decreased by HILI overexpression, overexpression of TBCB inhibited the increase of acetylation level of α-tubulin induced by HILI knockdown. (E) Immunofluorescence assays showed that HILI down-regulated acetylation level of α-tubulin in a TBCB-dependent manner (a, NC; b, HILI; c, sh-TBCB; d, HILI/sh-TBCB; e, sh-HILI; f, TBCB; g, sh-HILI/TBCB; AC, AC-α-tubulin).

Mentions: Acetylated α-tubulin can only be detected in polymerized microtubules333435. To prove whether there is a relationship between HILI and microtubule, we detected the change of acetylation level of α-tubulin and α-tubulin expression altered by HILI. Western blotting analyses showed that HILI overexpression decreased acetylation level of α-tubulin and HILI knockdown increased acetylation level of α-tubulin, but α-tubulin expression was not significantly changed in HeLa and HepG2 cells (Fig. 1A). Laser confocal microscopy (LSCM) was also introduced to detect the acetylation level of α-tubulin, showing that up-regulation of HILI decreased acetylation level of α-tubulin and down-regulation of HILI increased acetylation level of α-tubulin in HeLa and HepG2 cells, but fluorescence intensity of α-tubulin had no significant change (Fig. 1B). To further prove that HILI inhibits microtubule polymerization, a tubulin polymerization in vivo assay was performed, showing that HILI overexpression significantly decreased polymerized α-tubulin and HILI knockdown significantly increased polymerized α-tubulin in HeLa and HepG2 cells (Fig. 1C). We further studied how HILI inhibited microtubule polymerization.When TBCB was overexpressed simultaneously, HILI knockdown can no longer increase the acetylation level of α-tubulin and vice versa (Fig. 1D). Similar results were also observed in immunofluorescence experiments (Fig. 1E). These results suggested that HILI inhibits microtubule polymerization in a TBCB-dependent manner.


HILI destabilizes microtubules by suppressing phosphorylation and Gigaxonin-mediated degradation of TBCB
HILI suppresses microtubule polymerization in a TBCB-dependent manner.(A) HILI down-regulated acetylation level of α-tubulin and had no significant effect on α-tubulin expression at protein level in HeLa and HepG2 cells. HeLa and HepG2 cells were transfected with MYC-HILI, sh-NC, or sh-HILI vector. After 48 h, cell lysates were prepared for Western blotting with AC-α-tubulin and α-tubulin antibody (AC, AC-α-tubulin). (B) Immunofluorescent staining of AC-α-tubulin and α-tubulin in transfected cells. (C) In vivo tubulin polymerization assays in HeLa and HepG2 cells. Supernatant (S) and pellet (P) fractions of cell lysates were analyzed with anti-α-tubulin, data were presented as mean ± sd. (*P < 0.05). (D) Knockdown of TBCB recovered acetylation level of α-tubulin decreased by HILI overexpression, overexpression of TBCB inhibited the increase of acetylation level of α-tubulin induced by HILI knockdown. (E) Immunofluorescence assays showed that HILI down-regulated acetylation level of α-tubulin in a TBCB-dependent manner (a, NC; b, HILI; c, sh-TBCB; d, HILI/sh-TBCB; e, sh-HILI; f, TBCB; g, sh-HILI/TBCB; AC, AC-α-tubulin).
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f1: HILI suppresses microtubule polymerization in a TBCB-dependent manner.(A) HILI down-regulated acetylation level of α-tubulin and had no significant effect on α-tubulin expression at protein level in HeLa and HepG2 cells. HeLa and HepG2 cells were transfected with MYC-HILI, sh-NC, or sh-HILI vector. After 48 h, cell lysates were prepared for Western blotting with AC-α-tubulin and α-tubulin antibody (AC, AC-α-tubulin). (B) Immunofluorescent staining of AC-α-tubulin and α-tubulin in transfected cells. (C) In vivo tubulin polymerization assays in HeLa and HepG2 cells. Supernatant (S) and pellet (P) fractions of cell lysates were analyzed with anti-α-tubulin, data were presented as mean ± sd. (*P < 0.05). (D) Knockdown of TBCB recovered acetylation level of α-tubulin decreased by HILI overexpression, overexpression of TBCB inhibited the increase of acetylation level of α-tubulin induced by HILI knockdown. (E) Immunofluorescence assays showed that HILI down-regulated acetylation level of α-tubulin in a TBCB-dependent manner (a, NC; b, HILI; c, sh-TBCB; d, HILI/sh-TBCB; e, sh-HILI; f, TBCB; g, sh-HILI/TBCB; AC, AC-α-tubulin).
Mentions: Acetylated α-tubulin can only be detected in polymerized microtubules333435. To prove whether there is a relationship between HILI and microtubule, we detected the change of acetylation level of α-tubulin and α-tubulin expression altered by HILI. Western blotting analyses showed that HILI overexpression decreased acetylation level of α-tubulin and HILI knockdown increased acetylation level of α-tubulin, but α-tubulin expression was not significantly changed in HeLa and HepG2 cells (Fig. 1A). Laser confocal microscopy (LSCM) was also introduced to detect the acetylation level of α-tubulin, showing that up-regulation of HILI decreased acetylation level of α-tubulin and down-regulation of HILI increased acetylation level of α-tubulin in HeLa and HepG2 cells, but fluorescence intensity of α-tubulin had no significant change (Fig. 1B). To further prove that HILI inhibits microtubule polymerization, a tubulin polymerization in vivo assay was performed, showing that HILI overexpression significantly decreased polymerized α-tubulin and HILI knockdown significantly increased polymerized α-tubulin in HeLa and HepG2 cells (Fig. 1C). We further studied how HILI inhibited microtubule polymerization.When TBCB was overexpressed simultaneously, HILI knockdown can no longer increase the acetylation level of α-tubulin and vice versa (Fig. 1D). Similar results were also observed in immunofluorescence experiments (Fig. 1E). These results suggested that HILI inhibits microtubule polymerization in a TBCB-dependent manner.

View Article: PubMed Central - PubMed

ABSTRACT

Human PIWIL2, aka HILI, is a member of PIWI protein family and overexpresses in various tumors. However, the underlying mechanisms of HILI in tumorigenesis remain largely unknown. TBCB has a critical role in regulating microtubule dynamics and is overexpressed in many cancers. Here we report that HILI inhibits Gigaxonin-mediated TBCB ubiquitination and degradation by interacting with TBCB, promoting the binding between HSP90 and TBCB, and suppressing the interaction between Gigaxonin and TBCB. Meanwhile, HILI can also reduce phosphorylation level of TBCB induced by PAK1. Our results showed that HILI suppresses microtubule polymerization and promotes cell proliferation, migration and invasion via TBCB for the first time, revealing a novel mechanism for HILI in tumorigenesis.

No MeSH data available.