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. 2013 Mar;33(6):1114-23.
doi: 10.1128/MCB.01044-12. Epub 2012 Dec 28.

Tubulin acetyltransferase αTAT1 destabilizes microtubules independently of its acetylation activity

Nereo Kalebic  1 , Concepcion Martinez, Emerald Perlas, Philip Hublitz, Daniel Bilbao-Cortes, Karol Fiedorczuk, Annapaola Andolfo, Paul A Heppenstall
Affiliations

Tubulin acetyltransferase αTAT1 destabilizes microtubules independently of its acetylation activity

Nereo Kalebic et al. Mol Cell Biol. 2013 Mar.

Abstract

Acetylation of α-tubulin at lysine 40 (K40) is a well-conserved posttranslational modification that marks long-lived microtubules but has poorly understood functional significance. Recently, αTAT1, a member of the Gcn5-related N-acetyltransferase superfamily, has been identified as an α-tubulin acetyltransferase in ciliated organisms. Here, we explored the function of αTAT1 with the aim of understanding the consequences of αTAT1-mediated microtubule acetylation. We demonstrate that α-tubulin is the major target of αTAT1 but that αTAT1 also acetylates itself in a regulatory mechanism that is required for effective modification of tubulin. We further show that in mammalian cells, αTAT1 promotes microtubule destabilization and accelerates microtubule dynamics. Intriguingly, this effect persists in an αTAT1 mutant with no acetyltransferase activity, suggesting that interaction of αTAT1 with microtubules, rather than acetylation per se, is the critical factor regulating microtubule stability. Our data demonstrate that αTAT1 has cellular functions that extend beyond its classical enzymatic activity as an α-tubulin acetyltransferase.

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Figures

Fig 1
Fig 1
Expression pattern of αTAT1 mRNA. (A) Five different αTAT1 transcripts (see Fig. S1 in the supplemental material) were analyzed by quantitative real-time PCR (n = 3). All transcripts were expressed at low levels in all tested tissues (DRG, brain, spinal [Sp.] cord, heart, kidney, lungs, and liver). The error bars indicate standard errors. (B to I) In situ hybridization for αTAT1. (B) E16.5 mice show strong αTAT1 mRNA expression signal in the central nervous system. (D to H) Adult mice display expression in the nervous system, especially in the hippocampus (D), cortex (E), cerebellum (F) and DRG (H), and also in the testis (G). (C and I) Control hybridizations with sense probe. Bars, 100 μm (B and C), 500 μm (D, E, and F), 50 μm (G), and 40 μm (H and I).
Fig 2
Fig 2
αTAT1 colocalizes and interacts with microtubules. (A and B) Endogenously expressed αTAT1 in DRG neurons colocalizes with acetylated α-tubulin, especially in axons (B). (C) Transfected CHO cells show that αTAT1-YFP is recruited to the mitotic spindle during cell division. The cells were costained with an antibody against α-tubulin. Endogenous levels of αTAT1 were too low to be detected with the anti-αTAT1 antibody. (D) GST pulldown assay showing interaction between bacterially expressed αTAT1-GST and purified bovine microtubules. Ten percent of the input was loaded, and the membrane was blotted against α-tubulin. Scale bars, 20 μm (A) and 5 μm (B and C).
Fig 3
Fig 3
Tubulin and αTAT1 itself are the primary substrates of αTAT1. (A) αTAT1-mediated acetylation predominates at microtubules. CHO cells were transfected with YFP-αTAT1 and stained with panspecific antiacetyllysine antibody and α-tubulin antibody. Scale bar, 20 μm. (B) αTAT1-YFP transfection increases cytoplasmic acetylation, as determined from fluorescence intensity measurements of acetyllysine staining (P < 0.01; n > 10). (C) Transfected CHO whole-cell extracts immunoblotted with panspecific antiacetyllysine antibody showing that tubulin is the major target of αTAT1. (D) Acetyllysine immunoblot of CHO cells treated with the histone deacetylase inhibitor TSA (5 μM) for 4 h. αTAT1-overexpressing cells display increased tubulin acetylation and an additional band (arrow) that corresponds to αTAT1. (E) Autoradiography of αTAT1-mediated [14C]acetyl incorporation into polymerized microtubules (MT). In the absence of tubulin, autoacetylation is more evident. (F) Concentration dependence of αTAT1-mediated acetylation of microtubules (n = 3). (G) αTAT1 preferentially acetylates polymerized tubulin. Paclitaxel and Polymerized, microtubules treated with 3 μM paclitaxel or 1 mM GTP plus 15% glycerol, respectively, for 30 min before the acetylation reaction; Polymerizing, microtubules polymerized with 1 mM GTP and 15% glycerol during the acetylation reaction; Soluble, microtubules incubated with 1 mM GDP and without glycerol during acetylation. The error bars indicate standard errors.
Fig 4
Fig 4
Autoacetylation increases the catalytic activity of αTAT1. (A and B) Autoradiography of [14C]acetyl incorporation into αTAT1. Recombinant αTAT1 is present as two bands (37 kDa and 30 kDa). For both bands, saturation of the autoacetylation reaction is achieved after 24 h (n = 3). (C and D) Preincubation of αTAT1 with [14C]acetyl-CoA for 24 h increases its subsequent catalytic activity at tubulin (n = 3; P < 0.02). (E and F) αTAT1 autoacetylation regulates its catalytic activity in vivo. (E) Immunoblot of mock-, αTAT1-, and αTAT1-4R-transfected NIH 3T3 cell extracts against acetylated tubulin and actin (n = 3). (F) Quantification of acetylated tubulin (n = 3; P < 0.02). The error bars indicate standard errors.
Fig 5
Fig 5
αTAT1 decreases resistance to nocodazole. (A) NIH 3T3 cells were transfected with αTAT1 shRNA (KD), scrambled shRNA (SC), or αTAT1-YFP (OE). The insets show transfected cells. Twenty-four hours posttransfection, nocodazole was applied for the indicated time. Cells with αTAT1 knockdown show increased resistance on nocodazole. (B) Quantification of data from panel A. Polymerized, cells with an intact MT network; Fragmented and Depolymerized, partial and widespread depolymerization, respectively.
Fig 6
Fig 6
αTAT1 reduces levels of detyrosinated tubulin. (A to D) NIH 3T3 cells, transfected with mock YFP (A), αTAT1 shRNA (B), αTAT1-YFP (C), and catalytically inactive αTAT1-GGL-YFP (D), were stained with anti-detyrosinated tubulin antibody. The αTAT1- and αTAT1-GGL-transfected cells (the insets show transfected cells) show reduced detyrosination. (E) The proportion of cells with stable microtubules is increased upon transfection with αTAT1 shRNA and reduced after transfection with αTAT1 and the catalytically inactive mutant αTAT1-GGL (n > 100). Scale bars, 20 μm.
Fig 7
Fig 7
αTAT1 increases microtubule dynamics. (A to F) Live-cell images with a color-coded overlay representing EB3 particle growth speed. NIH 3T3 cells were transfected with TagRFP-EB3 and mock YFP (A), αTAT1 shRNA (B), YFP-αTAT1 (C), and catalytically inactive YFP-αTAT1-GGL (D). Video microscopy data were analyzed using the plusTipTracker software package. (E and F) Data for the speed of plus-tip growth (E) and for particle displacement (F) (n = 20). (G to J) Live-cell imaging with TagRFP–α-tubulin. Microtubule dynamicity (the sum of all growth events and all shortening events divided by total time) (G) and the percentage of time microtubules spend in growth or shortening (H) are increased upon YFP-αTAT1 and YFP-αTAT1-GGL overexpression and decreased upon αTAT1 knockdown (the numbers indicate percentages). (I and J) Data for the speed of shortening and frequency of catastrophe (the number of transitions from growth and pause to shortening per minute). The error bars indicate standard errors.
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