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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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Model for regulation of axonemal tubulin acetylation in the primary cilium by PP1 and I-2. Axonemal tubulin acetylation is regulated by the balance of acetyltransferase (HAT) and deacetylases (HDACs). The tubulin-localized HDAC binds and interacts with MT-associated PP1. The activity of HDAC is negatively regulated by phosphorylation, so PP1 dephosphorylation produces activation. The presence of I-2 inhibits this PP1 holoenzyme and keeps the PP1-bound HDAC inactive. As a result the axonemal tubulin is highly acetylated and stabilized during formation of the primary cilium. When I-2 is depleted or dissociated from PP1-HDAC complex, PP1 dephosphorylates and activates HDAC, and results in ciliary tubulin deacetylation and destabilization of the axoneme of the primary cilium. Inhibition of PP1 by calyculin A or inhibition of HDAC by TSA can compensate for the function of I-2 in this pathway to increase tubulin acetylation.
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Figure 9: Model for regulation of axonemal tubulin acetylation in the primary cilium by PP1 and I-2. Axonemal tubulin acetylation is regulated by the balance of acetyltransferase (HAT) and deacetylases (HDACs). The tubulin-localized HDAC binds and interacts with MT-associated PP1. The activity of HDAC is negatively regulated by phosphorylation, so PP1 dephosphorylation produces activation. The presence of I-2 inhibits this PP1 holoenzyme and keeps the PP1-bound HDAC inactive. As a result the axonemal tubulin is highly acetylated and stabilized during formation of the primary cilium. When I-2 is depleted or dissociated from PP1-HDAC complex, PP1 dephosphorylates and activates HDAC, and results in ciliary tubulin deacetylation and destabilization of the axoneme of the primary cilium. Inhibition of PP1 by calyculin A or inhibition of HDAC by TSA can compensate for the function of I-2 in this pathway to increase tubulin acetylation.

Mentions: Taken in sum, our data suggest a model (Fig. 9) for the regulation of tubulin acetylation by I-2 in the primary cilium of human epithelial cells. Axonemal tubulin acetylation is a balance between tubulin acetyltransferase and deacetylase. We propose that a HDAC forms a complex with a PP1 holoenzyme that binds I-2. I-2 inhibits the PP1 activity, keeping HDAC in an inactive, phosphorylated state. When I-2 is knocked down or dissociated from the PP1-HDAC complex, an increase of PP1 activity leads to dephosphorylation and activation of HDAC, favoring deacetylation of tubulin and destabilization of the primary cilium axoneme. This model invokes phosphorylation to negatively regulate HDAC-6, but the opposite has been suggested, i.e. HDAC-6 activation by phosphorylation. An alternative could be regulation of HDAC-6 by binding to PP1 with I-2 acting as an allosteric modifier of PP1 inhibition of HDAC-6. More detailed study of HDAC6-PP1 complexes should distinguish between these models.


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

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

Model for regulation of axonemal tubulin acetylation in the primary cilium by PP1 and I-2. Axonemal tubulin acetylation is regulated by the balance of acetyltransferase (HAT) and deacetylases (HDACs). The tubulin-localized HDAC binds and interacts with MT-associated PP1. The activity of HDAC is negatively regulated by phosphorylation, so PP1 dephosphorylation produces activation. The presence of I-2 inhibits this PP1 holoenzyme and keeps the PP1-bound HDAC inactive. As a result the axonemal tubulin is highly acetylated and stabilized during formation of the primary cilium. When I-2 is depleted or dissociated from PP1-HDAC complex, PP1 dephosphorylates and activates HDAC, and results in ciliary tubulin deacetylation and destabilization of the axoneme of the primary cilium. Inhibition of PP1 by calyculin A or inhibition of HDAC by TSA can compensate for the function of I-2 in this pathway to increase tubulin acetylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Model for regulation of axonemal tubulin acetylation in the primary cilium by PP1 and I-2. Axonemal tubulin acetylation is regulated by the balance of acetyltransferase (HAT) and deacetylases (HDACs). The tubulin-localized HDAC binds and interacts with MT-associated PP1. The activity of HDAC is negatively regulated by phosphorylation, so PP1 dephosphorylation produces activation. The presence of I-2 inhibits this PP1 holoenzyme and keeps the PP1-bound HDAC inactive. As a result the axonemal tubulin is highly acetylated and stabilized during formation of the primary cilium. When I-2 is depleted or dissociated from PP1-HDAC complex, PP1 dephosphorylates and activates HDAC, and results in ciliary tubulin deacetylation and destabilization of the axoneme of the primary cilium. Inhibition of PP1 by calyculin A or inhibition of HDAC by TSA can compensate for the function of I-2 in this pathway to increase tubulin acetylation.
Mentions: Taken in sum, our data suggest a model (Fig. 9) for the regulation of tubulin acetylation by I-2 in the primary cilium of human epithelial cells. Axonemal tubulin acetylation is a balance between tubulin acetyltransferase and deacetylase. We propose that a HDAC forms a complex with a PP1 holoenzyme that binds I-2. I-2 inhibits the PP1 activity, keeping HDAC in an inactive, phosphorylated state. When I-2 is knocked down or dissociated from the PP1-HDAC complex, an increase of PP1 activity leads to dephosphorylation and activation of HDAC, favoring deacetylation of tubulin and destabilization of the primary cilium axoneme. This model invokes phosphorylation to negatively regulate HDAC-6, but the opposite has been suggested, i.e. HDAC-6 activation by phosphorylation. An alternative could be regulation of HDAC-6 by binding to PP1 with I-2 acting as an allosteric modifier of PP1 inhibition of HDAC-6. More detailed study of HDAC6-PP1 complexes should distinguish between these models.

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