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Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells.

Wang W, Brautigan DL - BMC Cell Biol. (2008)

Bottom Line: Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation.There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes.Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cell Signaling, University of Virginia, School of Medicine, Charlottesville, 22908, USA.

ABSTRACT

Background: Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood.

Results: Endogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium.

Conclusion: The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium.

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Isolation and analysis of cilia fraction from ARPE-19 cells. (A) Western blot to show levels of acetylated tubulin, I-2, PP1C and actin in equal numbers of sub-confluent and confluent cells used to isolate a cilia fraction. (B) Sub-confluent and 48 hr post confluent cells were used in parallel for cilia isolation by Calcium Shock and ultracentrifugation. Equal volumes of the cilia fraction were analyzed by SDS-PAGE in 12% gels stained with Commassie. (C) Western blotting to show the presence of I-2, PP1C and acetylated tubulin in the cilia fraction of sub-confluent cells and cells harvested 48 hrs after confluence.
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Figure 4: Isolation and analysis of cilia fraction from ARPE-19 cells. (A) Western blot to show levels of acetylated tubulin, I-2, PP1C and actin in equal numbers of sub-confluent and confluent cells used to isolate a cilia fraction. (B) Sub-confluent and 48 hr post confluent cells were used in parallel for cilia isolation by Calcium Shock and ultracentrifugation. Equal volumes of the cilia fraction were analyzed by SDS-PAGE in 12% gels stained with Commassie. (C) Western blotting to show the presence of I-2, PP1C and acetylated tubulin in the cilia fraction of sub-confluent cells and cells harvested 48 hrs after confluence.

Mentions: We confirmed the localization of endogenous I-2 in the primary cilium by immunoblotting analysis of a cilia fraction dissociated from cells using the calcium shock method and recovered by differential ultracentrifugation. Equivalent numbers of sub-confluent and post-confluent cells were used as starting material in the cilia isolation procedure. Immunoblotting showed that there were identical amounts of actin and PP1 in these samples (Fig. 4A). Post-confluent cells had slightly lower levels of acetylated tubulin and I-2 compared to sub-confluent cells (Fig. 4A). The cilia fractions obtained from sub-confluent and post-confluent cells were resolved by SDS-PAGE, and proteins stained by Coomassie (Fig. 4B). Relatively few proteins were recovered in the cilium fraction from sub-confluent cells, whereas post-confluent cells yielded about a dozen of prominent proteins of different sizes. Proteins recovered in this cilia fraction represented selected enrichment of a subset of the proteins present in a whole extract of ARPE-19 cells. Immunoblotting identified acetylated tubulin, PP1C and I-2 in the cilia fraction from post-confluent cells that were not in the cilia fraction of sub-confluent cells (Fig. 4C). These results showed that acetylated tubulin, I-2 and PP1C were recovered in the primary cilium by biochemical fractionation, which reinforces the immunostaining of I-2 in the primary cilium of individual cells.


Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells.

Wang W, Brautigan DL - BMC Cell Biol. (2008)

Isolation and analysis of cilia fraction from ARPE-19 cells. (A) Western blot to show levels of acetylated tubulin, I-2, PP1C and actin in equal numbers of sub-confluent and confluent cells used to isolate a cilia fraction. (B) Sub-confluent and 48 hr post confluent cells were used in parallel for cilia isolation by Calcium Shock and ultracentrifugation. Equal volumes of the cilia fraction were analyzed by SDS-PAGE in 12% gels stained with Commassie. (C) Western blotting to show the presence of I-2, PP1C and acetylated tubulin in the cilia fraction of sub-confluent cells and cells harvested 48 hrs after confluence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2630314&req=5

Figure 4: Isolation and analysis of cilia fraction from ARPE-19 cells. (A) Western blot to show levels of acetylated tubulin, I-2, PP1C and actin in equal numbers of sub-confluent and confluent cells used to isolate a cilia fraction. (B) Sub-confluent and 48 hr post confluent cells were used in parallel for cilia isolation by Calcium Shock and ultracentrifugation. Equal volumes of the cilia fraction were analyzed by SDS-PAGE in 12% gels stained with Commassie. (C) Western blotting to show the presence of I-2, PP1C and acetylated tubulin in the cilia fraction of sub-confluent cells and cells harvested 48 hrs after confluence.
Mentions: We confirmed the localization of endogenous I-2 in the primary cilium by immunoblotting analysis of a cilia fraction dissociated from cells using the calcium shock method and recovered by differential ultracentrifugation. Equivalent numbers of sub-confluent and post-confluent cells were used as starting material in the cilia isolation procedure. Immunoblotting showed that there were identical amounts of actin and PP1 in these samples (Fig. 4A). Post-confluent cells had slightly lower levels of acetylated tubulin and I-2 compared to sub-confluent cells (Fig. 4A). The cilia fractions obtained from sub-confluent and post-confluent cells were resolved by SDS-PAGE, and proteins stained by Coomassie (Fig. 4B). Relatively few proteins were recovered in the cilium fraction from sub-confluent cells, whereas post-confluent cells yielded about a dozen of prominent proteins of different sizes. Proteins recovered in this cilia fraction represented selected enrichment of a subset of the proteins present in a whole extract of ARPE-19 cells. Immunoblotting identified acetylated tubulin, PP1C and I-2 in the cilia fraction from post-confluent cells that were not in the cilia fraction of sub-confluent cells (Fig. 4C). These results showed that acetylated tubulin, I-2 and PP1C were recovered in the primary cilium by biochemical fractionation, which reinforces the immunostaining of I-2 in the primary cilium of individual cells.

Bottom Line: Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation.There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes.Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cell Signaling, University of Virginia, School of Medicine, Charlottesville, 22908, USA.

ABSTRACT

Background: Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood.

Results: Endogenous phosphatase inhibitor-2 (I-2) was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium.

Conclusion: The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium.

Show MeSH
Related in: MedlinePlus