# A phospho-regulated ensemble signal motif of α-TAT1 drives dynamic microtubule acetylation

Abhijit Deb Roy, Evan Gross, Gayatri Pillai, Shailaja Seetharaman, Sandrine

Etienne-Manneville, Takanari Inoue

► To cite this version:

Abhijit Deb Roy, Evan Gross, Gayatri Pillai, Shailaja Seetharaman, Sandrine Etienne-Manneville, et al.. A phospho-regulated ensemble signal motif of α-TAT1 drives dynamic microtubule acetylation. 2021. pasteur-03096546

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Preprint submitted on 5 Jan 2021

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1 A phospho-regulated ensemble signal motif of α-TAT1 drives dynamic microtubule

2 acetylation

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5 Abhijit Deb Roy¹*, Evan G. Gross², Gayatri S. Pillai², Shailaja Seetharaman³,⁴, Sandrine

6 Etienne-Manneville³, Takanari Inoue¹*

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8 ¹Department of Cell Biology and Center for Cell Dynamics, Johns Hopkins University School

9 of Medicine, 855 North Wolfe Street, Baltimore, MD 21205, USA

10 ²The Johns Hopkins University, Baltimore, MD 21218, USA

11 ³Cell Polarity, Migration and Cancer Unit, Institut Pasteur, UMR3691 CNRS, Equipe

12 Labellisée Ligue Contre le Cancer, F-75015, Paris, France.

13 ⁴Université Paris Descartes, Sorbonne Paris Cité, 12 Rue de l'École de Médecine, 75006

14 Paris, France.

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16 * Correspondence:

17 Abhijit Deb Roy abhijit.debroy@gmail.com

18 Takanari Inoue jctinoue@jhmi.edu

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20 Abstract:

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**Introduction:**

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Acetylation of Lysine-40 of α-tubulin is an evolutionarily conserved post-translational modification observed across eukaryotic species¹⁻³, which is involved in diverse physiological and pathological states⁴⁻⁶. Acetylation is mainly observed in polymerized microtubules⁷⁻⁹ and may provide structural flexibility to stabilize microtubules against bending forces¹⁰⁻¹³. In cultured cells, microtubule acetylation mediates focal adhesion dynamics, adaptation to extracellular matrix rigidity as well as regulation of tissue stiffness¹⁴⁻¹⁷. Additionally, acetylated microtubules regulate touch sensation in *M. musculus*, *C. elegans* and *D. melanogaster*, suggesting a role in mechano-response¹⁸⁻²¹. Microtubule acetylation has been implicated in axonal transport in neurons²²⁻²⁴, migration in cancer cells⁵,²⁵⁻²⁷, autophagy⁶,²⁸,²⁹, podosome stabilization in osteoclasts³⁰ and viral infections³¹⁻³⁴. α-TAT1 is the only known acetyltransferase for α-tubulin³⁵,³⁶ in mammals. α-TAT1 predominantly catalyzes α-tubulin in stable polymerized microtubules¹⁹,³⁷, and may have additional effects on microtubules independent of its catalytic activity³⁸.

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Although microtubule acetylation is spatially and temporally regulated downstream of many molecular signaling pathways, little is known about how these pathways converge on α-TAT1 to achieve such dynamic patterns. In the present study, we used computational sequence analyses and live cell microscopy to identify a conserved motif in the intrinsically disordered C-terminus of α-TAT1, consisting of an NES and an NLS, that mediates its spatial distribution. We show that cytosolic localization of α-TAT1 is critical for microtubule acetylation. We further demonstrate that nuclear localization of α-TAT1 is inhibited by the action of serine-threonine kinases, specifically cyclin dependent kinases (CDKs), Protein Kinase A (PKA) and Casein kinase 2 (CK2) and identify 14-3-3 proteins as binding partners of α-TAT1. Our findings establish a novel role of the intrinsically disordered C-terminus in
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controlling α-TAT1 function by regulating its intracellular localization downstream of kinase

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and phosphatase activities.

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65 **Results:**

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67 **α-TAT1 localization mediates microtubule acetylation:** α-TAT1 has an N-terminal catalytic domain that shows homology to other acetyltransferases, while its C-terminus was not resolved in crystal structures³⁹ (Fig. 1a). Based on its amino acid sequence, α-TAT1 C-terminus was predicted to be intrinsically disordered by both IUPred2A⁴⁰ and PrDOS⁴¹ prediction servers (Fig. 1a, Supplementary Fig. S1a, b, c). To explore if the intra-cellular localization of α-TAT1 is regulated, we sought to identify any localization signals present in the α-TAT1 amino acid sequence (Supplementary Fig. S1a). The prediction program NetNES⁴² identified a putative NES in α-TAT1 C-terminus (Fig. 1a, Supplementary Fig. S2). On the other hand, PSORT-II⁴³ subcellular localization program predicted that α-TAT1 should be predominantly localized to the nucleus due to the presence of a putative class 4 NLS⁴⁴ in its C-terminus (Fig. 1a). The region encompassing the putative NES and NLS is conserved across the human α-TAT1 isoforms (Supplementary Fig. S3a), as well as across mammalian α-TAT1 proteins (Supplementary Fig. S3b). To test whether α-TAT1 indeed showed any intracellular distribution pattern, we expressed mVenus-α-TAT1 in HeLa cells, and observed distinct nuclear exclusion in most cells, although a subset of cells showed lack of exclusion (Fig. 1b). Based on our categorization of the distribution patterns (see Methods, Fig. 1c), we determined that approximately 77% of cells showed cytosolic distribution, 22% showed diffused pattern (i.e., both cytosolic and nuclear distribution) and 1% of cells showed nuclear enrichment of mVenus-α-TAT1 (Fig. 1d). These observations were consistent with ratiometric analysis (see Methods, Fig 1c) of mVenus-α-TAT1 distribution (Fig. 1e). Time lapse microscopy showed temporal changes in fluorescence intensity of mVenus-α-TAT1 in cell nuclei and cytosol (Supplementary Fig. S4a, b), suggesting that α-TAT1 localization is dynamic by nature.
90 α-TAT1 preferentially acetylates polymerized microtubules, which are typically 91 cytosolic. Based on this, we hypothesized that spatial regulation of α-TAT1 may control its 92 function. Exogenous expression of mVenus-α-TAT1 or its catalytic domain (residues 1- 93 236)¹⁹,³⁹ (Fig. 1f) was sufficient to significantly increase α-tubulin acetylation in HeLa cells 94 compared to non-transfected cells (Fig. 1g, h). To test whether nuclear localization may 95 sufficiently sequester α-TAT1 from microtubules, we tethered the NLS from cMyc to α-TAT1 96 catalytic domain and thus localized it to the nucleus (Fig. 1f, g top panel). Exogenous 97 expression of NLS-mVenus-α-TAT1(1-236) did not increase α-tubulin acetylation levels 98 compared to non-transfected cells (Fig. 1 g, h), suggesting that nuclear sequestration of α- 99 TAT1 inhibits its function. 100

101 α-TAT1 undergoes Exportin 1 dependent nuclear export: To dissect the molecular 102 mechanisms of α-TAT1 localization in the cytosol, we speculated that α-TAT1 is actively 103 exported out of the nucleus, and/or that α-TAT1 binds to a protein that keeps the complex out 104 of the nucleus. We began by testing the first possibility by assessing involvement of the 105 nuclear export machinery. Exportin 1 (Exp1), also called Chromosome region maintenance 1 106 protein homolog (CRM1), mediates nuclear export of many proteins⁴⁵,⁴⁶. GFP-α-TAT1, but not 107 GFP, co-immunoprecipitated with endogenous Exp1 (Fig. 2a), indicating an interaction 108 between these two proteins. Treatment with 100 nM Leptomycin-B (LMB), an inhibitor of Exp1 109 mediated nuclear export⁴⁷, significantly decreased the number of cells displaying nuclear 110 exclusion of mVenus-α-TAT1 compared to vehicle (Fig 2b, c, d). Inhibition of nuclear export 111 was initiated within an hour of LMB treatment, although some cells were refractory to the 112 treatment (Supplementary Fig. S5a, b). Decreased LMB concentrations (1 nM and 10 nM) 113 had a comparable impact as 100 nM dosage (Supplementary Fig. S5c, d). Furthermore, LMB 114 treatment induced significant reduction in α-tubulin acetylation levels in HeLa cells within 4
hours, compared to vehicle (Fig. 2e, f). Our data suggest that α-TAT1 is actively exported
from the nucleus to the cytosol in an Exp1 dependent manner and that this export facilitates
α-TAT1 function. The residual exclusion of α-TAT1 could be due to Exp1-independent nuclear
export pathways or association with other cytosolic proteins, which was tested later (see
below).

Nuclear export of α-TAT1 is mediated by a C-terminal NES: Our data demonstrate that α-TAT1 function is linked to Exp1 mediated nuclear export. To examine whether nuclear export of α-TAT1 is regulated by its catalytic activity, we expressed a catalytic dead mutant, mVenus-α-TAT1(D157N)³⁹, in HeLa cells. mVenus-α-TAT1(D157N) did not display any loss of nuclear exclusion (Supplementary Fig. S6a, b, c). The catalytic domain, mVenus-α-TAT1(1-236), displayed a complete loss of nuclear exclusion; whereas the C-terminus, mVenus-α-TAT1(236-323), displayed a distribution pattern comparable to that of WT (Fig. 3a, b, c, d). In addition, inhibition of Exp1 by 100 nM LMB significantly reduced the nuclear exclusion of mVenus-α-TAT1 C-terminus (Fig. 3e). Exclusion of α-TAT1 C-terminus (size ≈ 38 kDa) further indicates that nuclear exclusion of mVenus-α-TAT1 (size ≈ 63 kDa) is not simply due to size exclusion of passive diffusion into nuclei, and demonstrates that nuclear exclusion of α-TAT1 is a transferable property mediated by its C-terminus.

Exp1 dependent nuclear export is typically mediated by binding with short stretches of
hydrophobic, often Leucine rich, NES⁴²,⁴⁵ that are often found in disordered regions of the
cargo proteins. As previously mentioned, NetNES suggested the presence of a conserved
NES between V286 and L297 in α-TAT1 C-terminus (Supplementary Fig S2). Interestingly,
this region is also predicted to be a site of protein-protein interactions by ANCHOR2
prediction software⁴⁰ (Supplementary Fig. S1b). Truncation of this putative NES, α-TAT1-
delNES, abrogated its nuclear exclusion (Fig. 3a, b, c, d). Although alanine substitution of the
hydrophobic residues in the V286-L297 region, α-TAT1(VL/A), decreased its nuclear exclusion, the loss of exclusion was less than expected from our observations with LMB treatment (Supplementary Fig. S6a, b, c). On further examination of the NetNES prediction, we observed that the NES predicted by the Hidden Markov Model (Supplementary Fig. S2) included the residues between L282 and L297. Additional Alanine substitutions of L282 and F285, α-TAT1(NES/A) (Supplementary Fig. S6d), further reduced nuclear exclusion of α-TAT1 (Fig. 3a, b, c, d, Supplementary Fig. S6a, b, c), suggesting that these residues contribute to the nuclear export of α-TAT1. Taken together, our data suggest that α-TAT1 has a hydrophobic NES in its C-terminus.

**α-TAT1 interacts with 14-3-3 proteins.** Our observations thus far with nuclear exclusion of mVenus-α-TAT1 indicate that the putative NLS identified by the PSORT prediction server is either non-functional or is basally inhibited with occasional activation. 14-3-3 protein binding has been reported to negatively regulate nuclear import by inhibiting binding of importins to NLS⁴⁸⁻⁵⁰. Part of the putative NLS sequence “PAQRRRTR” bears similarity to 14-3-3 binding motif RXX(pS/pT)XP⁵¹, and 14-3-3-Pred⁵², a 14-3-3 interaction prediction server identified T322 as a potential 14-3-3 binding site. To examine whether α-TAT1 bound to 14-3-3 proteins, we co-expressed GFP-α-TAT1 with HA-tagged 14-3-3-β or 14-3-3-ζ in HEK-293T cells and performed a co-immunoprecipitation assay. GFP-α-TAT1, but not GFP alone, co-precipitated with HA-14-3-3-β and HA-14-3-3-ζ (Fig. 4a). These observations are consistent with our observations in mass spectrometry analysis of α-TAT1 in HEK cells that identified 14-3-3-β and 14-3-3-ζ as potential interactors⁵³.

**Serine/threonine kinase activities mediate cytosolic localization of α-TAT1.** 14-3-3β binding to proteins is mediated by phosphorylated serine and threonine residues⁵¹. NetPhos
165 prediction server identified over 30 putative phosphosites in α-TAT1, of which nine residues 166 have been reported to be phosphorylated in phospho-proteomic studies (Supplementary Fig. 167 S7a, b). Treatment with 100 nM Staurosporin, a pan-kinase inhibitor, significantly increased 168 nuclear localization of α-TAT1 (Fig. 4b, c, Supplementary Fig. S8), suggesting that 169 phosphorylation negatively regulated nuclear localization of α-TAT1. To identify the specific 170 kinases that regulate α-TAT1 localization, we treated cells expressing mVenus-α-TAT1 with 171 several serine-threonine kinase inhibitors (summarized in Supplementary Table T1). Both RO- 172 3306 (inhibitor for CDK1 and CDK2<sup>54,55</sup>) and PD0332991 (also called Pablociclib, inhibitor for 173 CDK4 and CDK6<sup>56,57</sup>), but not Purvalanol-B (inhibits CDK1, CDK2 and CDK5<sup>55</sup>), increased 174 nuclear localization of mVenus-α-TAT1 in HeLa cells (Fig. 4b, c, Supplementary Fig. S8). 175 Silmitasertib (also called CX-4945, inhibitor for CK2<sup>58</sup>) but not D-4476 (inhibitor for CK1<sup>59</sup>) 176 also had a similar impact (Fig. 4b, c, Supplementary Fig. S8). Although H-89 (inhibitor for 177 PKA) only had a moderate effect on mVenus-α-TAT1 localization, co-expression of mCFP- 178 PKI, a more potent inhibitor for PKA<sup>60</sup>, considerably increased nuclear localization of mVenus- 179 α-TAT1 (Fig 4c, Supplementary Fig. S8, Supplementary Table T1). Finally, treatment with 180 LJI308 (inhibitor for Ribosomal S9 kinase) appeared to further increase nuclear exclusion of 181 mVenus-α-TAT1 (Fig. 4c, Supplementary Table T1). These data demonstrate that nuclear 182 localization of α-TAT1 is negatively regulated by CDKs, CK2 and PKA. 183

184 **Nuclear localization of α-TAT1 is phospho-inhibited:** Since α-TAT1(1-284) did not display 185 nuclear exclusion (Fig. 3b, c, d), we reasoned that the phosphosites which inhibit nuclear 186 localization of α-TAT1 might be located between F285 and R323. NetPhos prediction server 187 identified S294, T303, S315 and T322 as potential phosphosites in this region 188 (Supplementary Fig. S7a, indicated in red box), wherein only S315 and T322 have been 189 reported to be phosphorylated in phosphoproteomic studies (Supplementary Fig. S7b) and
they also flank the putative NLS (Fig. 5a). Importin-α binds with NLS enriched in basic residues through a charge-based interaction⁴⁴. Phosphorylation of amino acids adjacent to such an NLS may inhibit the association of Importin-α binding through a disruption of the charge balance in the NLS region⁶¹. Alanine substitution of T322, but not of S315, significantly increased nuclear localization of mVenus-α-TAT1 (Fig. 5a, b, c, d, Supplementary Fig. S9a, b, c). Alanine substitution of both S315 and T322, α-TAT1(ST/A), showed considerably more nuclear localization of mVenus-α-TAT1 than T322 alone (Fig 5a, b, c, d). These data suggest that T322 phosphorylation inhibits nuclear localization of α-TAT1, while S315 may play a co-operative role in such inhibition. Substitution of S315 with acidic residues (S315D) appeared to boost nuclear exclusion of α-TAT1, whereas substitution of T322 with acidic residues (T322E) or both (ST/DE) displayed increased diffused pattern, but not increased nuclear accumulation (Supplementary Fig. S9a, b, c). This may be because these acidic residues, unlike phosphate moieties, do not sufficiently counter the basic residues in the NLS; or that phospho-T322, and to a lesser extent, phospho-S315 phosphorylation may be involved in protein-protein interactions, possibly with 14-3-3 isoforms, that inhibit nuclear localization of α-TAT1. These observations suggest that the phosphate moiety in phosphorylated T322 and S315 is critical for inhibition of α-TAT1 nuclear import.

One possible explanation of increased nuclear localization of T322A mutant is that phospho-T322 mediates nuclear export of α-TAT1. Truncation of the putative NLS including T322, α-TAT1-delNLS, did not increase nuclear localization (Fig. 5a, b, c, d), indicating that T322 did not mediate nuclear export of α-TAT1. Furthermore, alanine substitution of the hydrophobic residues in α-TAT1 NES as well as S315 and T322, α-TAT1(NES/A, ST/A) not only abrogated nuclear exclusion, but considerably increased nuclear accumulation of α-TAT1 (Fig. 5a, b, c, d), suggesting additive effects of NES inhibition and NLS activation. α-TAT1(NES/A, ST/DE) mutant also showed diffused pattern but not nuclear accumulation
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One of the bottlenecks in elucidating the role of microtubule acetylation in biological phenomena is the knowledge gap of how upstream molecular signaling pathways control α-TAT1 function to modulate microtubule acetylation. Our study demonstrates that intracellular α-TAT1 localization is a dynamically regulated process, orchestrated by a balance of nuclear export and import, which modulates microtubule acetylation levels (Fig. 5e). To our knowledge, this is the first study to identify the molecular mechanisms that spatially regulate α-TAT1. We have demonstrated a hitherto unknown role of the inherently disordered α-TAT1 C-terminus and identified novel interactions with 14-3-3 proteins and several kinases. TAK1 dependent phosphorylation of α-TAT1 Serine-237 has been reported to stimulate its catalytic property<sup>62</sup>. In neurons, p27<sup>kip1</sup> directly binds to α-TAT1 and stabilizes it against proteasomal degradation<sup>24</sup>, thus enhancing α-tubulin acetylation. Our observation that spatial sequestration of α-TAT1 from microtubules modulates acetylation dynamics suggests a role of the nucleus as a reservoir or sequestration chamber to control protein access of substrates. Regulated spatial sequestration of biomolecules can control their action<sup>63-67</sup> and aberrant localization of proteins have been reported in many diseases<sup>68-71</sup>. Our study further highlights the role of spatial signaling processes in controlling protein function.

We have demonstrated active nuclear export of α-TAT1 by Exp1 through an NES rich in hydrophobic residues, which was critical for efficient microtubule acetylation. In addition, we have identified an NLS consistent with non-canonical class IV NLS<sup>44</sup>. Interestingly, position 7 of this NLS, which should not be an acidic residue, is occupied by Threonine-322. Since phosphorylation of threonine can significantly increase its net negative charges, it is ideally situated to act as an ON/OFF switch for the NLS. Although we have identified Threonine-322 to be the critical phospho-residue that inhibits nuclear import, Serine-315 appears to provide
243 additional inhibition. The increased nuclear localization of ST/A mutant over T322A mutant
244 raises the possibility that S315 and T322 may aggregate signals from different signaling
245 pathways to fine-tune α-TAT1 localization.

246 Our data demonstrate that nuclear localization of α-TAT1 is inhibited by kinase action,
247 possibly on Threonine-322 and Serine-315. Specifically, our study shows a role of CDKs, PKA
248 and CK2 in coordinating spatial distribution of α-TAT1. Such phospho-regulation of α-TAT1
249 provides a possible mechanism for the changes in α-TAT1 localization and microtubule
250 acetylation observed at different stages of the cell cycle²². We identified Threonine-322 to be
251 a putative binding site for 14-3-3 proteins and demonstrated that α-TAT1 binds to 14-3-3-β
252 and 14-3-3-ζ proteins. 14-3-3s typically interact with phospho-serines or phospho-threonines
253 in intrinsically disordered regions and may mediate nuclear transport of proteins by masking
254 NES or NLS²³. Furthermore, 14-3-3 proteins may significantly alter the structure of their
255 binding partners to align along their rigid α-helical backbone, to expose or hide critical binding
256 sites²⁴. Comparable kinase-mediated regulation of nuclear export and nuclear import has
257 previously been reported in a few transcription regulators⁷⁵⁻⁷⁸. In particular, regulation of
258 Cdc25 localization by Checkpoint kinase1 (Chk1) mediated phosphorylation of and
259 subsequent recruitment of 14-3-3-β to an NLS-proximal phosphosite is virtually the same as
260 our proposed model (Fig. 5e) of α-TAT1 localization⁷⁸,⁷⁹, suggesting that such kinase-
261 mediated balancing of nuclear export and import is a general strategy for protein localization.
262 α-TAT1 is unique in this aspect in that unlike the other proteins that are spatially regulated in
263 this manner, α-TAT1 has no known substrates in the nucleus and that its nuclear localization
264 appears to be primarily to sequester it from microtubules. Of course, it is possible that nuclear
265 import of α-TAT1 facilitates interactions with presently unidentified substrates located in the
266 nucleus. In a similar vein, 14-3-3 proteins and Exp1 are also acetylated⁸⁰,⁸¹, and it is intriguing
267 to speculate that these might be substrates of α-TAT1.
It is worthwhile to consider that a significant number of post-translational modifications of α-TAT1 appear on its intrinsically disordered C-terminus (Supplementary Fig. 7b). Disordered regions may act as a signaling hub by interacting with multiple proteins, thus facilitating complex formation and acting as integrators of signaling pathways<sup>82</sup>. We have demonstrated the presence of an NES, NLS, phosphorylation sites and putative 14-3-3 binding sites within the α-TAT1 C-terminus. That it is well conserved across mammalian species as well as in all the human isoforms suggests a critical role of the α-TAT1 C-terminus in its function. Indeed, numerous cancer-associated mutations curated in COSMIC<sup>83</sup> (www.cancer.sanger.ac.uk) and TCGA Research Network (www.cancer.gov/tcga) databases, in the ATAT1 gene are located in the intrinsically disordered C-terminal region. More specifically, there are a considerable number of deletions, frame shifts and missense mutations encompassing the NES and the NLS regions, which may be expected to affect the spatial distribution of α-TAT1. Whether these mutations underlie the pathogenesis in these cancers remain to be examined. Considering the role of microtubule acetylation in a wide array of cellular activities, it may be conjectured that loss of spatial regulation of α-TAT1 may be present in other diseases as well.

In conclusion, we propose a new model for regulation of microtubule acetylation through spatial sequestration of α-TAT1 (Fig. 5e), which include three key aspects: presence of an NES that facilitates Exp1 mediated nuclear export, presence of an NLS to mediate nuclear import and finally, modulation of this nuclear import by kinases. Further investigation into the role of specific kinases on α-TAT1 localization may yield a better understanding of its function in cellular processes and pathologies and help identify new therapeutic targets.
290 **Materials and Methods**

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292 **Cell culture and transfection:** HeLa and HEK-293T cells were cultured in DMEM basal
293 media and passaged every third day of culture. For optimal growth, the media were
294 supplemented with 10% (v/v) fetal bovine serum and Penicillin/Streptomycin, and the cells
295 were maintained under standard cell culture conditions (37 °C and 5% CO2). The cell lines
296 were regularly checked for mycoplasma contamination. FuGENE 6 reagent (Promega,
297 Madison, WI) was used for transient transfection of HeLa cells according to the
298 manufacturer's instructions. For immunoprecipitation assays, HEK cells were transfected
299 using calcium phosphate method.

300 **DNA plasmids:** H2B-mCherry construct was a generous gift from Dr. Sergi Regot. α-TAT1
301 construct was a generous gift from Dr. Antonina Roll-Mecak. The α-TAT1 construct was
302 subcloned into the pTriEx-4 vector (Novagen) using PCR and restriction digestion with
303 mVenus at the N terminus and α-TAT1 at the C terminus. H2B-mCherry and CFP-PKI
304 constructs were respectively subcloned into mCherry-C1 and mCer3-C1 vectors (Clontech).
305 GFP-αTAT1 construct was a gift from Dr. Philippe Chavrier and Dr. Guillaume
306 Montagnac. HA-14-3-3 plasmids were a generous gift from Dr. Michael Yaffe. As indicated in
307 the results and figure legends, tags of compatible fluorescent proteins including Cerulean,
308 mVenus and mCherry were appended to facilitate detection. Unless specified otherwise, the
309 termini of tagging were positioned as in the orders they were written. Truncations of α-TAT1
310 were generated by PCR. Point mutations of α-TAT1 were generated using overlapping PCR.
311 The open reading frames of all DNA plasmids were verified by Sanger sequencing.

312 **Sequence alignment:** Protein sequence alignment was performed using Clustal-W<sup>84</sup>
313 (https://www.ebi.ac.uk/Tools/msa/clustalo/).
**Nuclear transport and kinase inhibitors:** LMB was purchased from LC Laboratories (catalog # L6100). SB203580 (Sigma Aldrich, catalog # S8307), Doramapimod (BIRB 796, Selleck Chemicals, catalog # S1574), CHIR99021 (Sigma Aldrich, catalog # SML1046), Sostrastaurin (Selleck Chemicals, catalog # S2791), RO-3306 (Selleck Chemicals, catalog # S7747), Ipatasertib (RG7440, Selleck Chemicals, catalog # S2808), Capivasertib (AZD5363, Selleck Chemicals, catalog # S8019), Silmitasertib (CX 4945, Selleck Chemicals, catalog # S2248), KU-55933 (Sigma Aldrich, catalog # SML1109) were generous gifts from Dr. Sergi Regot. SB239063 (Sigma Aldrich, catalog # S0569) was a generous gift from Dr. Jun Liu. The rest of the kinase inhibitors were purchased as indicated: Staurosporine (Sigma Aldrich, catalog # 569397), LJ1308 (Sigma Aldrich, catalog # SML1788), Y-27632 (LC Laboratories, catalog # Y-5301), Gö 6976 (Sigma Aldrich, catalog # 365250), Gö 6983 (Sigma Aldrich, catalog # G1918), H-89 (Sigma Aldrich, catalog # B1427), D4476 (BioVision, catalog # 1770), KN-62 (Selleck Chemicals, catalog # S7422), KU-57788 (MedChemExpress, catalog # HY-11006), Purvalanol B (AdipoGen Life Sciences, catalog # SYN-1070), KT-5823 (Cayman Chemicals, catalog # 10010965), PD0332991 (Sigma Aldrich, catalog # PZ0199).

**Immunofluorescence assays:** HeLa cells were transiently transfected with mVenus-α-TAT1, mVenus-α-TAT1 catalytic domain and NLS-mVenus-α-TAT1 catalytic domain. 24 hours post-transfection, cells were fixed using ice-cold methanol for 10 minutes, washed thrice with cold PBS, blocked with 1% BSA in PBS for one hour and then incubated overnight at 4°C with monoclonal antibodies against tubulin (Millipore, catalog # MAB1864) and acetylated α-Tubulin (Sigma Aldrich, catalog # T7451). Next day, the samples were washed thrice with cold PBS and incubated with secondary antibodies (Invitrogen) for one hour at room temperature, after which they were washed thrice with PBS and images were captured by microscopy. For LMB treatment, HeLa cells were dosed with 100 nM LMB or equal volume of vehicle (EtOH),
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incubated for 4 hours, after which methanol fixation and immunostaining was performed as
described above.

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**Immunoprecipitation assays:** HEK293T cells were transiently transfected with pEGFP-c1 (GFP-Ctl) or GFP-αTAT1 with HA-14-3-3β or HA-14-3-3ζ using the calcium phosphate method. Cell lysates were prepared by scraping cells using 1X lysis buffer (10X recipe - 50 mM Tris pH 7.5, triton 20%, NP40 10%, 2 M NaCl, mixed with cOmplete protease inhibitor tablet - Roche, Product number 11873580001). Cell lysates rotated on a wheel at 4°C for 15 min and centrifuged for 10 min at 13,000 rpm 4°C to pellet the cell debris. A small volume of the supernatant was used as the soluble input. Soluble detergent extracts were incubated with GFP nanobody (NanoTag, N0310) for 1 h at 4°C. Samples were then centrifuged and washed thrice with wash buffer (250 mM NaCl, 0.1% Triton X-100 in PBS). The resin and the soluble input were then mixed with Laemmli buffer (composed of 60 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS and 50 mM DTT with the addition of protease and phosphatase inhibitors). Samples were boiled 5 min at 95°C before loading in polyacrylamide gels. Gels were transferred for western blot and membranes were blocked with TBST (0.1% Tween) and 5% milk and incubated 1 h with the primary antibody and 1 h with HRP-conjugated secondary antibody. Bands were revealed with ECL chemiluminescent substrate (Biorad). Two different western blots were used to visualize GFP-Input and HA-14-3-3 proteins due to similar molecular weights. Antibodies used: GFP-HRP (NB600-313, Novus Biologicals), anti-HA (rat; Merck; 11867423001), anti-exportin-1 (mouse; BD Transduction Laboratories™; 611832). Secondary HRP antibodies were all purchased from Jackson ImmunoResearch.

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**Microscopy and image analyses:** All imaging was performed with an Eclipse Ti microscope (Nikon) with a 100X objective (1.0X zoom and 4X4 binning) and Zyla 4.2 sCMOS camera (Andor), driven by NIS Elements software (Nikon). Time lapse imaging was performed at 15 min intervals for 10-15 hours. All live cell imaging was conducted at 37°C, 5% CO₂ and 90%

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humidity with a stage top incubation system (Tokai Hit). Vitamin and phenol red-free media (US Biological) supplemented with 2% fetal bovine serum were used in imaging to reduce background and photobleaching. Inhibitors and vehicles were present in the imaging media during imaging. All image processing and analyses were performed using Metamorph (Molecular Devices, Sunnyvale, CA, USA) and FIJI software (NIH, Bethesda, MD, USA).

For categorical analysis of mVenus-α-TAT1 localization, images were visually inspected and classified as displaying either cytosolic, diffused, or nuclear localization of mVenus fluorescence signal. For ratiometric analysis, the ratio of the fluorescence intensity from region of interest (≈ 10 µm diameter) in the nucleus to that in a perinuclear area was used to minimize any volumetric artifacts (Fig. 1c). To determine the baseline Nuc/cyto ratio for cytosolic (<0.8) and nuclear (>1.2), we visually identified WT mVenus-α-TAT1 cells, with or without LMB treatment, that showed distinctly cytosolic or nuclear localization and used the rounded average ratio values from these cells (n > 100 cells). All cells showing nuc/cyto ratio in between were classified as diffused. For both categorical and ratiometric analyses, H2B-mCherry signal was used to identify nuclei; in a few cases where H2B-mCherry signal was absent, phase images were used to identify the nuclei. Cells displaying too much (near saturation in 16 bit) or too little (approximately less than 1.5-fold signal over background) of mVenus or mCherry fluorescence signal, or those which appeared to be dying on visual inspection were not included during image acquisition.

For immunofluorescence assays with exogenous expression of mVenus-α-TAT1 plasmids, transfected cells were identified by the presence of mVenus fluorescence signal. The ratio of acetylated α-Tubulin over α-Tubulin (Ac. α-Tub/α-Tub) for transfected cells was normalized against that for non-transfected cells averaged over 20 untransfected cells from the same dish. For LMB and vehicle treatment, Ac. α-Tub/α-Tub ratios are shown.
**Statistical analyses:** Microsoft Excel (Microsoft, Redmond, WA, USA) and R (R Foundation for Statistical Computing, Vienna, Austria) were used for statistical analyses. Exact number of samples for each data set are specified in the respective figure legends. Data was pooled from at least three independent experiments (technical replicates) and within each experiment typically data from at least 40 cells were obtained (biological replicates). In some cases with kinase inhibitors that induced cell death, we collected data from smaller number of cells per experiment but increased the number of experiments to ensure sufficient data. Sample sizes were chosen based on the commonly used range in the field without performing any statistical power analysis. Normal probability plot (Supplementary Fig. S10) was utilized to confirm normal distribution of the Nuc/Cyto ratio of mVenus-αTAT1. Extreme outliers (<Q1 – 3xIQR or >Q3+3xIQR) were excluded from plots and statistical analyses. *P*-values were obtained from two-tailed Students *t*-test assuming unequal variance. Exact *P*-values for kinase inhibitor as-says are available in Supplementary Table T1.

**Data availability:** All relevant data and source codes are included. Plasmid constructs will be available through Addgene.
402 Acknowledgements

403 We thank Allen Kim for discussions that led to initiation of this project and Amy F. Peterson for
404 help with the kinase inhibitor assays. We thank Yuta Nihongaki and Helen D. Wu for
405 constructive discussions, as well as Robert DeRose for proofreading. This project was
406 supported by American Heart Association fellowship 20POST35220046 (ADR), discretionary
407 funds (TI), the La Ligue contre le cancer (S-CR17017) and Centre National de la Recherche
408 Scientifique and Institut Pasteur. SS is funded by the ITN PolarNet Marie Curie grant and
409 Fondation pour la Recherche Médicale and is enrolled at the Ecole Doctorale Frontières du
410 Vivant (FdV) – Programme Bettencourt.

411

412 Contributions

413 ADR initiated the project and designed and performed most of the experiments and data
414 analyses. GSP and EGG performed experiments and data analyses under the guidance of
415 ADR and TI. SS performed the immunoprecipitation assays under the guidance of SEM. ADR
416 and TI wrote the final version of the manuscript based on contributions from all the authors.

417

418 Competing interests

419 The authors declare no competing interests.

420
421 **Figure legends**

422 **Figure 1. Intracellular distribution of α-TAT1 mediates its function.**

423 a) Cartoon showing predicted NES and NLS in intrinsically disordered C-terminus of α-TAT1, 424 adapted from PDB: 4GS4, b) intracellular distribution of mVenus-α-TAT1, red dotted lines out- 425 line nuclei as identified by H2B-mCherry in lower panel, c) cartoon showing criteria for cate- 426 gorical and ratiometric analyses of α-TAT1 distribution, d) categorical analysis (n = 1032 cells) 427 and e) ratiometric analysis (n = 304 cells) of mVenus-α-TAT1 localization, f) cartoon showing 428 α-TAT1 mutants used in g) immunofluorescence assays showing levels of acetylated α-tubulin 429 and total α-tubulin, transfected cells are indicated with red arrowheads, h) ratio of acetylated 430 α-tubulin to total tubulin intensities with exogenous expression of α-TAT1 and its mutants, 431 normalized against that of non-transfected cells, (WT: 50, catalytic domain: 44, NLS-catalytic 432 domain: 48 cells). Scale bar = 10µm. **": P<0.001 and NS: not significant, Student's t-test.** 433

434

435 **Figure 2. α-TAT1 undergoes Exp1 mediated nuclear export.**

436 a) Co-immunoprecipitation of endogenous Exp1 with GFP-α-TAT1, b) intracellular distribution 437 of mVenus-α-TAT1 with vehicle (EtOH) and 100 nM LMB treatment, nuclei are indicated in red 438 c) categorical analysis (WT: 1032, vehicle: 450, LMB: 495 cells) and d) rati- 439 ometric analysis (WT: 304, vehicle: 210, LMB: 240 cells) of mVenus-α-TAT1 localization with 440 vehicle and LMB treatment, e) immunofluorescence images showing acetylated and total α- 441 tubulin in HeLa cells with vehicle or LMB treatment, f) ratio of acetylated to total α-tubulin with 442 vehicle or LMB treatment (vehicle:120, LMB: 130 cells). Scale bar = 10µm. **": P<0.001 and 443 NS: not significant, Student's t-test.** 444

444

444 **Figure 3. Intracellular distribution of α-TAT1 is mediated by its C-terminus.**
a) Cartoon showing α-TAT1 mutant design, b) categorical analysis (WT: 1032, Cat. Dom: 371, C-term: 290, delNES: 343 and NES/A: 501 cells) and c) ratiometric analysis (WT: 304, Cat. Dom: 213, C-term: 220, delNES: 221 and NES/A: 212 cells) of mVenus-α-TAT1 mutant localization as indicated, d) representative images showing intracellular distribution of mVenus-α-TAT1 mutants as listed, red dotted lines outline nuclei, e) ratiometric analysis of intracellular distribution of mVenus-α-TAT1 C-term with 100 nM LMB (C-term: 220, LMB: 209 cells. Scale bar = 10µm. ***: P<0.001 and NS: not significant, Student's t-test.

452

Figure 4. Nuclear localization of α-TAT1 is phospho-inhibited.

a) Co-immunoprecipitation of GFP-α-TAT1 with HA-14-3-3β and HA-14-3-3ζ proteins, b) intracellular distribution of mVenus-α-TAT1 with Staurosporine, RO-3306, PD0332991 and Silmitasertib treatment, nuclei are indicated in red dotted lines, c) ratiometric analysis of mVenus-α-TAT1 localization with kinase inhibitors (WT:304, STS: 183, RO-3306: 221, PD0332991: 234, Purvalanol-B: 243, D4476:253, Silmitasertib: 253, H89: 219, PKI: 208, Ipatasertib: 259, Capivsertib: 218, Go6976: 297, Go6983: 241, Sostrastaurin: 401, KT-5823: 269, SB203580: 259, Birb796: 262, KU55933: 317, LJI308: 229, CHIR99021: 227, KN-62:210, KU-57788: 249, Y-27632: 161 cells). Scale bar = 10µm. ***: P<0.001 and NS: not significant, Student's t-test.

463

Figure 5. α-TAT1 has a C-terminal phospho-inhibited NLS.

a) Cartoon showing α-TAT1 putative NLS flanked by potential phosphosites and corresponding mutants, b) intracellular distribution of mVenus-α-TAT1 mutants as indicated, c) outlined in red dotted lines, d) ratiometric analysis (WT: 304, T322A: 228, ST/A: 194, delNLS: 208, NES/A, ST/A: 225 cells) and e) categorical analysis (WT: 1032, T322A: 346, ST/A: 290, delNLS: 563, NES/A, ST/A: 476 cells) of intracellular localization of mVenus-α-TAT1 mutants,

465

466

467

468

469
470 e) proposed model of α-TAT1 localization and its impact on microtubule acetylation. Scale bar

471 = 10µm. ***P<0.001 and NS, not significant, Student's t-test.

472
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<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>
bioRxiv preprint doi: https://doi.org/10.1101/2020.09.23.310235; this version posted December 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

<figure><img src="image_6.png" /><figcaption>Figure 5</figcaption></figure>

<figure><img src="image_7.png" /></figure>
**Supplementary Information**

**A phospho-regulated signal motif of α-TAT1 drives dynamic microtubule acetylation**

Abhijit Deb Roy¹*, Evan G. Gross², Gayatri S. Pillai², Shailaja Seetharaman³,⁴, Sandrine

Etienne-Manneville³, Takanari Inoue¹*

¹ Department of Cell Biology and Center for Cell Dynamics, Johns Hopkins University School

of Medicine, Baltimore, 855 North Wolfe Street, MD 21205, USA

² The Johns Hopkins University, Baltimore, MD 21218, USA

³Cell Polarity, Migration and Cancer Unit, Institut Pasteur, UMR3691 CNRS, Equipe

Labellisée Ligue Contre le Cancer, F-75015, Paris, France.

⁴Université Paris Descartes, Sorbonne Paris Cité, 12 Rue de l'École de Médecine, 75006

Paris, France.

* Correspondence:

Abhijit Deb Roy abhijit.debroy@gmail.com

Takanari Inoue jctinoue@jhmi.edu
a) Query: α-TAT1 Isoform 7
MEFPFDVDALFPERITVLDQHLRPPARRPGTTTPARVDLQQQIMTIIDELGKASAKAQNLSAPITSA
SRMQSNRHVVYILKDSSARPAGKGAIIGFIKVGYKKLFVLDDREAHNEVEPLCILDFYIHESVQRH
GHGRELFQYMLQKERVEPHQLAIDRPSQKLLKFLNKHYNLETTVPQVNNFVIFEGFFAHQHRPPA
PSLRATRHSRAAAVDPTPAAPARKLPPKRAEGDIKPYSSSDREFLKVAVEPPWPLNRAPRRATP
PAHPPPRSSSLGNSPERGPLRPFVPEQELLRSLRLCPPHPTARLLLAADPGGSPAQRRRT³²²R

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Supplementary Figure S1.** αTAT1 C-terminus is disordered. a) amino acid sequence of αTAT1 isoform 7 used as the query, the putative NES and NLS are in bold, b) IUPred2 (and ANCHOR2) and c) PrDOS results suggesting that αTAT1 C-terminus is disordered. The threshold to be considered disordered is 0.5 for both.</figcaption></figure>
<figure><img src="image_10.png" /></figure>

Hidden Makov Model (HMM) algorithm prediction: **LRPFVPEQELLRSRLRL**

**Supplementary Figure S2.** NetNES prediction for NES in αTAT1 identifying the putative NES, the predicted NES from NetNES optimized algorithm (pink) and that from Hidden Markov Model (blue) are shown with the hydrophobic residues indicated in bold.
<figure><img src="image_11.png" /><figcaption>**Supplementary Figure S3.** The putative NES and NLS in α-TAT1 are evolutionarily conserved. a) Alignment of human α-TAT1 isoforms, b) alignment of different mammalian α-TAT1 showing the C-terminal region including the putative NES and NLS (enclosed in boxes), genus of the mammalian α-TAT1 are shown. Alignments were performed using ClustalW (https://www.ebi.ac.uk/Tools/msa/clustalo/)</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Supplementary Figure S4.** α-TAT1 intracellular localization is dynamic in nature. a) Spontaneous temporal changes in spatial distribution of mVenus-α-TAT1, scale bar = 10 µm b) temporal changes in nuclear fluorescence intensity of mVenus-α-TAT1 in HeLa cells, each line indicates normalized nuclear intensity of mVenus-α-TAT1 in a single cell, n = 20 cells</figcaption></figure>
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Supplementary Figure S5.** α-TAT1 undergoes CRM1 dependent nuclear export. a), b) Temporal changes in mVenus-α-TAT1 localization on 100 nM LMB treatment after LMB addition at time 0, scale bar = 10 µm, for b), each line indicates normalized nuclear intensity of mVenus-α-TAT1 in a single cell, black line shows the mean and gray line indicate the 95% C.I., n = 26 cells , c) categorical analysis (WT: 1032, vehicle: 450, 1 nM: 329, 10 nM: 291, 100 nM: 495 cells) and d) ratiometric analysis (WT: 304, vehicle: 210, 1 nM: 203, 10 nM: 200, 100 nM: 240 cells) of mVenus-α-TAT1 localization on vehicle (EtOH) or LMB treatment at 1, 10 and 100 nM.</figcaption></figure>

***P<0.001 and NS, not significant, Student's t-test.
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

**Supplementary Figure S6.** α-TAT1 has a C-terminal NES. a) α-TAT1 NES predicted by NetNES and mutants using alanine substitution, b) categorical (WT: 1032, D157N: 429, LMB: 495, VL/A: 436, NES/A: 501 cells), and c) ratiometric (WT: 304, D157N: 217, LMB: 240, VL/A: 201, NES/A: 212 cells) analyses of intracellular localization of mVenus-α-TAT1 and mutants as indicated.

***P<0.001 and NS, not significant, Student's t-test.
<figure><img src="image_20.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_21.png" /></figure>

**Supplementary Figure S7.** a) Putative phosphosites in α-TAT1 predicted by NetPhos, those in F285-R323 are highlighted, b) currently reported post-translational modifications in α-TAT1 curated by PhosphoSitePlus®, www.phosphosite.org.
<figure><img src="image_22.png" /></figure>

**Supplementary Figure S8.** Categorical analysis of α-TAT1 localization on treatment with kinase inhibitors. WT: 1032, STS: 157, R0-3306: 796, PD0332991: 543, Purvalanol B: 992, D4476: 689, 418, H89: 251, PKI: 354, Ipatasertib: 546, Capivasertib: 385, Go6976: 712, Go6983: 606, Sostrastaurin: 605, KT5823: 570, SB203580: 505, Birb796: 991, KU55933: 815, LJI308: 522, CHIR99021: 484, KN62: 531, KU57788: 949, Y27632: 340 cells.
<figure><img src="image_23.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_25.png" /></figure>

**Supplementary Figure S9.** α-TAT1 has a C-terminal phospho-inhibited NLS. a) Cartoon showing α-TAT1 NES and NLS flanked by potential phosphosites and mutants, b) categorical (WT:1032, S315A: 442, S315D: 412, T322E: 310, ST/DE: 258 cells) and c) ratiometric (WT: 304, S315A: 212, S315D: 223, T322E: 232, ST/DE: 195 cells) analyses of intracellular localization of mVenus-α-TAT1 mutants. ***P<0.001 and NS, not significant, Student's t-test.
<figure><img src="image_26.png" /><figcaption>**Supplementary Figure S10.** Normal probability plot of nuclear/cytosolic ratio of mVenus-α-TAT1 expressed in HeLa cells.</figcaption></figure>
<table><thead><tr><th>Drug</th><th>Conc. Used</th><th>target kinase</th><th>Incubation time</th><th>p value (t-test)</th></tr></thead><tbody><tr><td>STS</td><td>100 nM</td><td>Pan kinase</td><td>4 hours</td><td>1.20187E-11</td></tr><tr><td>RO3306</td><td>10 µM</td><td>Cdk1, Cdk2</td><td>4 hours</td><td>1.87845E-10</td></tr><tr><td>PD0332991</td><td>1 µM</td><td>Cdk4, Cdk6</td><td>4 hours</td><td>1.29985E-10</td></tr><tr><td>Purvalanol B</td><td>1 µM</td><td>Cdk1, Cdk2, Cdk5</td><td>4 hours</td><td>0.086698844</td></tr><tr><td>D4476</td><td>1 µM</td><td>CK1</td><td>4 hours</td><td>0.244864719</td></tr><tr><td>Silmitasertib</td><td>10 µM</td><td>CK2</td><td>4 hours</td><td>7.34743E-12</td></tr><tr><td>H89</td><td>10 µM</td><td>PKA</td><td>4 hours</td><td>0.001868139</td></tr><tr><td>PKI</td><td>NA</td><td>PKA</td><td>Overnight</td><td>8.76993E-08</td></tr><tr><td>Ipatasertib</td><td>10 µM</td><td>PKB (Akt)</td><td>4 hours</td><td>0.026614985</td></tr><tr><td>Capivasertib</td><td>1 µM</td><td>PKB (Akt)</td><td>4 hours</td><td>0.928873535</td></tr><tr><td>Go6976</td><td>250 nM</td><td>PKC</td><td>4 hours</td><td>0.900843024</td></tr><tr><td>Go6983</td><td>250 nM</td><td>PKC</td><td>4 hours</td><td>0.068144954</td></tr><tr><td>Sostrastaurin</td><td>10 µM</td><td>PKC</td><td>4 hours</td><td>0.008916116</td></tr><tr><td>KT5823</td><td>5 µM</td><td>PKG</td><td>4 hours</td><td>0.581314971</td></tr><tr><td>SB203580</td><td>10 µM</td><td>p38 MAPK</td><td>4 hours</td><td>0.007945499</td></tr><tr><td>Birb796</td><td>10 µM</td><td>p38 MAPK</td><td>4 hours</td><td>0.768174955</td></tr><tr><td>KU55933</td><td>10 µM</td><td>ATM</td><td>4 hours</td><td>0.26994365</td></tr><tr><td>LJI308</td><td>10 µM</td><td>RSK</td><td>4 hours</td><td>9.62544E-07</td></tr><tr><td>CHIR99021</td><td>1 µM</td><td>GSK3</td><td>4 hours</td><td>0.309440886</td></tr><tr><td>KN62</td><td>10 µM</td><td>CaMK-II, P2RX7</td><td>4 hours</td><td>0.021582087</td></tr><tr><td>KU57788</td><td>1 µM</td><td>DNA-PK</td><td>4 hours</td><td>0.385979985</td></tr><tr><td>Y27632</td><td>10 µM</td><td>ROCK</td><td>4 hours</td><td>0.375473038</td></tr></tbody></table>

**Supplementary Table T1.** Details of kinase inhibitors used.