Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells
© Wang and Brautigan; licensee BioMed Central Ltd. 2008
Received: 26 August 2008
Accepted: 26 November 2008
Published: 26 November 2008
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.
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.
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.
Cilia are projections from the surface of cells that are similar to flagella. The axoneme of a primary cilium is made up of microtubules. Each cilium (and flagellum) grows out from, and remains attached to, a basal body, which is the maternal centriole . Almost every cell in vertebrates has a single primary cilium with a 9+0 arrangement , which lacks the central pair of microtubules seen in the 9+2 arrangement of flagella and motile cilia. Primary cilia are essential for several critical signaling pathways, sensory reception and detection of fluid flow across epithelia. Recent studies suggest that a variety of human syndromes are related to defects in the assembly, maintenance and function of the primary cilium, including renal dysfunction, diabetes, and retinal degeneration [3, 4].
Little is currently known about the control of the formation and resorption of cilia, although many proteins have been defined as ciliary structural components or cilia-associated signaling proteins. Several lines of evidence indicate a relationship between cell cycle and primary cilium assembly. Ciliary disassembly in many cells precedes entry into the cell cycle and ciliary assembly follows exit from mitosis [5, 6]. Some proteins, such as NIMA-related kinase  and Aurora kinase [8, 9], have been shown to play roles in both cell cycle regulation and assembly of cilia.
Protein phosphatase 1 (PP1) is a major protein Ser/Thr phosphatase with a variety of cellular functions [10–12]. PP1 exists in cells as a set of distinctive multisubunit holoenzymes [12, 13], which are comprised of a PP1 catalytic subunit paired with a regulatory subunit. There are predicted to be 200+ regulatory subunits that control PP1 holoenzyme subcellular localization, catalytic activity and substrate specificity. Protein phosphatase inhibitor-2 (I-2) is a heat stable protein capable of selectively inhibiting the PP1 catalytic subunit . Recent studies show that the function of I-2 is important for the cell cycle regulation and I-2 is localized at centrosomes during interphase . The expression level of I-2 fluctuates during the cell cycle and is enhanced at mitosis , when it becomes phosphorylated at PXTP site [17, 18]. Knockdown of I-2 by RNAi in mammalian cells leads to failure of cytokinesis and formation of multinucleated cells, probably due to an imbalance of Aurora B vs. PP1 . During Drosophila early embryogenesis, maternal I-2 is required for proper chromosome segregation and mitotic synchrony . The concept is that I-2 selectively targets certain PP1 holoenzymes to control kinase/PP1 balance and thereby trigger cellular events.
Here we report that endogenous I-2 is localized in the primary cilium of human retinal epithelial ARPE-19 cells. During the process of cilium formation, I-2 was concentrated in the cilium before axonemal tubulin was acetylated. Knockdown of I-2 by RNAi specifically reduced tubulin acetylation in the primary cilium, not the rest of the cell, and this could be rescued by chemical inhibition of either PP1 or HDAC. Our results indicate that I-2 has a role in regulation of tubulin acetylation in the primary cilium.
Localization of I-2 in the primary cilium of human retinal pigment epithelial cells
Induction of primary cilium formation in ARPE-19 cells
Detection of I-2 and PP1 in isolated primary cilia
I-2 concentrates in the primary cilium prior to tubulin acetylation
I-2 is required for robust acetylation of tubulin in the primary cilium
To demonstrate that the phenotype of reduced tubulin acetylation in the primary cilium was not due to the off-target effects of RNAi, we used another pair of siRNA targeting a different coding region of I-2. The knockdown efficiency and the phenotype were the same (data not shown). Alternatively, we combined siRNA transfection in sub-confluent ARPE-19 cells with induction of the primary cilium by serum starvation. In this protocol the primary cilium is formed without signals from cell-cell contacts that are formed during confluence. Knockdown of I-2 in sub-confluent cells significantly decreased the percentage of cells with a primary cilium and reduced tubulin acetylation in the primary cilium (not shown). Reduced tubulin acetylation by I-2 knockdown would be expected to reduce axonemal microtubule stability, and probably thereby result in the overall lower fraction of cells with a primary cilium.
Reversal of I-2 knockdown phenotypes by inhibition of HDAC or PP1
We sought to obtain stringent proof of the specificity of RNAi by rescue of the knockdown phenotypes by over-expression of I-2, using an expression vector mutated to avoid RNAi. We over-expressed silently mutated I-2 in ARPE-19 cells, but found this severely reduced the percentage of confluent cells that formed a primary cilium (not shown). Therefore, we had to adopt alternative approaches to rescue the reduced acetylation of tubulin phenotype in I-2 knockdown cells.
The other approach was to inhibit protein phosphatase type-1 (PP1) and type 2A (PP2A) using the cell-permeable compound calyculin A. Calyculin A is effective at nanomolar doses and shows some preference for PP2A over PP1 [21, 22]. We tested a range of concentrations from 0.1 to 2 nM, and found that at 0.5 nM calyculin A there was partial rescue of I-2 knockdown cells in the fraction of cells with a primary cilium and of tubulin acetylation in the primary cilium (Fig. 8). Treatment of cells with the same doses of TSA or calyculin A without knockdown of I-2 produced no significant change in the fraction of cells with a primary cilium, and no change in tubulin acetylation in the primary cilium (Fig. 8). These results suggest that partial inhibition of PP1 was able to reverse the effect of I-2 knockdown.
This study shows that phosphatase inhibitor 2 (I-2) is concentrated in the primary cilium of human epithelial cells. We found endogenous I-2 localizes to the primary cilium prior to acetylation of the axonemal tubulin that serves as a marker for the primary cilium. Knockdown of I-2 by siRNA significantly suppressed the full acetylation of tubulin in the axoneme of the primary cilium, and reduced the percentage of cells with a primary cilium. These effects were rescued by pharmacological inhibition of PP1 or HDACs. Our results link I-2 and PP1, proteins involved in control of protein Ser/Thr phosphorylation, to the acetylation of tubulin in the primary cilium. This links two different systems of protein post-translational modification in the primary cilium.
Tubulin acetylation and deacetylation are catalyzed by a tubulin acetyltransferase and a specific histone/tubulin deacetylase (HDAC). Acetylation stabilizes microtubules , and the modification has been mapped to a single residue K40 of alpha-tubulin [24, 25]. However, the identity of the acetyltransferase reactive with this site is still unknown. Among the multiple histone deacetylases, HDAC-6 has been shown to interact with and catalyze alpha tubulin K40 deacetylation [26, 27]. HDAC-6 has been proposed to destabilize microtubules of the axoneme in the primary cilium and be regulated through phosphorylation involving Aurora A . Previous results have shown that histone deacetylases, such as HDAC-1, HDAC-6, and HDAC-10 bind directly to the PP1 catalytic subunit [28, 29]. Inhibition of HDACs by TSA causes dissociation of these HDAC-PP1 complexes, presumably through a conformational change. . On the other hand, ATM dependent activation of PP1 by ionizing radiation led to dissociation of HDAC-PP1 complexes and dephosphorylation of HDAC-1, with an increase of HDAC activity . Phosphorylation of I-2 directly by ATM is proposed to cause dissociation from PP1, accounting for PP1 activation by ionizing radiation . Previous results therefore established functional links between PP1, HDAC-6 and I-2 and we suggest these are related to acetylation of tubulin in the primary cilium of epithelial cells.
PP1 was found in proteomic analysis of purified flagella from Chlamydomonas  and isolated human ciliary axonemes . This supports the idea that PP1 regulates Ser/Thr phosphorylation of proteins in flagella and cilia. In addition, RT-PCR analysis showed that the levels of PP1 mRNA increased by 1.9-fold upon deflagellation, considered as evidence that PP1 is involved in flagellum function in Chlamydomonas. PP1 co-purifies with microtubules (MT) and binding to microtubules is mediated by the MT-associated protein Tau . MT-associated phosphoproteins that might be PP1 substrates include kinesin and dynein motor complexes that are responsible for intraflagellar transport (IFT). However, the lack of change in size of the primary cilium in I-2 knockdown cells argues that I-2 does not regulate PP1 holoenzymes that control IFT. Altogether, various results point to some regulation of microtubule function and axoneme organization by PP1. In our experiments, inhibition of PP1 by 0.5 nM calyculin A rescued the reduction of alpha-tubulin acetylation in response to I-2 knockdown. Low doses of calyculin A are known to selectively inhibit PP2A [22, 34, 35], and we observed that they did not affect tubulin acetylation in the primary cilium in ARPE-19 cells (not shown). High doses of calyculin A were lethal to ARPE-19 cells. We surmised that the intermediate dose of calyculin A we used was producing selective but probably incomplete inhibition of PP1. We propose that I-2 affects tubulin acetylation and stabilization of the axoneme in the primary cilium by inhibiting the activity of specific PP1 holoenzymes. I-2 knockdown did not affect overall acetylation of tubulin, as detected by Western blotting whole cell extracts, or tubulin acetylation in either cytoplasm or midbody, as detected by immunostaining. We suspect that I-2 sensitive PP1 holoenzymes are specifically concentrated in the axoneme of the primary cilium to regulate ciliary alpha-tubulin acetylation, possibly by control of HDAC-6 activity. Previous work indicated that PP1 is targeted to microtubules by the protein Tau, however, Tau has not been identified by proteomics in flagella from Chlamydomonas or in axonemal fraction from cilia of human cells. This suggests that PP1 and therefore I-2 might be targeted to the primary cilium by some other PP1 regulatory subunit yet to be identified.
Lastly, we have demonstrated unanticipated multifunctionality of I-2 that will need to be reconciled into more complex models. I-2 is a mitotic phosphoprotein substrate of CDK1-cyclin B1 [17, 18]. In addition, I-2 binds and regulates the Pin1 prolyl isomerase . Both Nek2A and Aurora A kinases are activated by I-2, the former indirectly by PP1 inhibition, the latter directly by protein-protein interaction [37, 38]. In separate studies we have found that I-2 is required for proper chromosome segregation and cytokinesis, probably by indirect control of Aurora B . Thus, I-2 has proved to be quite a versatile protein. The primary cilium may take advantage of I-2 to connect and coordinate different signaling pathways.
Results of this study show that protein acetylation and phosphorylation are functionally interconnected to regulate the axonemal microtubules in the primary cilium. The regulatory protein I-2 localizes to the primary cilium prior to tubulin acetylation. Knockdown of I-2 by siRNA significantly reduced tubulin acetylation selectively in the primary cilium and chemical inhibitors of HDACs or PP1 partially reversed the effects of knockdown, showing that I-2 affects both phosphorylation and acetylation. As a mitotic phosphoprotein with multiple activities, I-2 may link cell cycle progression to the structure and function of the primary cilium.
Cell culture and transfection
Human adult retinal pigment epithelial cells (ARPE-19)(ATCC #CRL-2302) were grown according to ATCC recommendations. Cells were transfected with siRNA (80 nM) using Oligofectamine (Invitrogen) following the manufacturer' instructions. In the presence of serum, siRNA were incubated with cells 24 hrs prior to confluence to get efficient protein knockdown. Trichostatin A (TSA) was purchased from Sigma. Calyculin A was bought from Calbiochem. For the rescue assay, either 0.5 micromolar TSA or 0.5 nanomolar calyculin A was added into medium 24 hrs after siRNA transfection.
Two pairs of siRNA targeting different coding regions of I-2 were designed using Dharmacon program http://www.dharmacon.com/, and ordered from Dharmacon (Lafayette, Colorado). The sequences of siRNA are available upon request.
Western blotting as described  used the following primary antibodies: sheep polyclonal anti-I-2 (1:500); chicken anti-pan PP1 (1:20,000); mouse anti-actin (1:1000) (Sigma); mouse anti-acetylated tubulin (1:2000)(Sigma). Goat anti-rabbit Alexa Fluor 680, donkey anti-sheep Alexa Fluor 680 were purchased from Molecular Probes and Invitrogen and used at a 1:3000 dilution. Goat anti-mouse IRDye 800 and anti-chicken IRDye 800 antibodies were purchased from Rockland Immunochemicals and used at a 1:3000 dilution.
Immunofluorescent microscopy was done as described  using the following primary antibodies: Sheep polyclonal anti-I-2 (1:100); Mouse anti acetylated-tubulin (1:500) (Sigma); FITC-conjugated anti-alpha-tubulin; mouse anti gamma-tubulin (1:1000) (Sigma); mouse anti beta-tubulin (1:50) (DSHB). Rhodamine Red-X-conjugated goat anti-mouse, Oregon Green-conjugated goat anti-rabbit or goat anti-sheep secondary antibodies were used at 1:1000 (Molecular Probes). DNA was stained with Hoechst 33342. Wide field images were obtained using Nikon Eclipse E800 microscope equipped with a Hamamatsu 3580 camera using OpenLab software 3.0. Confocal images were obtained using an Olympus FluoView™ FV 1000 system.
Acetylated tubulin staining in cilia was analyzed with the image-analysis software Openlab® (Improvision, Coventry, UK). A mask (ROI) was created around acetylated tubulin staining in cilia. The mean pixel intensity of actylated tubulin staining in the masked region was corrected by subtracting background pixel intensity. A threshold of pixel intensity was set based on the comparison of staining intensity of control and I-2 knockdown cells. Axonemal tubulin in cilium whose acetylated tubulin staining intensity is above the threshold is counted as having normal tubulin acetylation, otherwise is counted as having inefficient tubulin acetylation.
Isolation of primary cilia fraction from ARPE19 cells
Primary cilia were isolated by Calcium Shock technique as described .
Protein phosphatase 1
This work was supported by grant from USPHS National Institute of Health GM-56362 to DLB. We appreciate encouragement by and discussions with Dr. Winfield S. Sale.
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