cells

Article HIPK2 Phosphorylates the -Severing Spastin at S268 for Abscission

1, , 1, 1 2 Alessandra Pisciottani † ‡, Loredana Biancolillo †, Manuela Ferrara , Davide Valente , Francesca Sardina 1, Laura Monteonofrio 2,§, Serena Camerini 3, Marco Crescenzi 3 , Silvia Soddu 2 and Cinzia Rinaldo 1,2,*

1 Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR), c/o Sapienza University, 00185 Rome, Italy 2 Unit of Cellular Networks and Molecular Therapeutic Targets; IRCCS-Regina Elena National Institute, 00144 Rome, Italy 3 Core Facilities, Italian National Institute of Health, 00161 Rome, Italy * Correspondence: [email protected] These Authors contributed equally to the work. † Present address: Vita-Salute San Raffaele University, 20132 Milan, Italy. ‡ § Present address: Laboratory of Cardiovascular Science, NIA/NIH Baltimore, MD 21224, USA.

 Received: 30 May 2019; Accepted: 3 July 2019; Published: 5 July 2019 

Abstract: Abscission is the final step of cell division, mediating the physical separation of the two daughter cells. A key player in this process is the microtubule-severing enzyme spastin that localizes at the midbody where its activity is crucial to cut and culminate the . Recently, we demonstrated that HIPK2, a multifunctional involved in several cellular pathways, contributes to abscission and prevents tetraploidization. Here, we show that HIPK2 binds and phosphorylates spastin at serine 268. During cytokinesis, the midbody-localized spastin is phosphorylated at S268 in HIPK2-proficient cells. In contrast, no spastin is detectable at the midbody in HIPK2-depleted cells. The non-phosphorylatable spastin-S268A mutant does not localize at the midbody and cannot rescue HIPK2-depleted cells from abscission defects. In contrast, the phosphomimetic spastin-S268D mutant localizes at the midbody and restores successful abscission in the HIPK2-depleted cells. These results show that spastin is a novel target of HIPK2 and that HIPK2-mediated of spastin contributes to its midbody localization for successful abscission.

Keywords: HIPK2; spastin; abscission; midbody; phosphorylation

1. Introduction Abscission is the final step of cytokinesis and consists of an orderly sequence of dynamic events that allow the final separation of the two nascent daughter cells, safeguarding the correct distribution of genomic and cytoplasmic materials in cell division [1,2]. At the end of cytokinesis, several membrane remodeling factors of the endosomal sorting complex required for transport-I (ESCRT-I) and -III pathways, such as Alix and CHMPs proteins, and the microtubule (MT)-severing enzyme spastin are sequentially recruited at the midbody, the organelle-like platform at the intercellular bridge [3]. In particular, CHMP1B recruits spastin, whose activity results in MT clearance leading to the physical separation of the two daughter cells [4–6]. This process depends on dramatic rearrangement of the cytoskeleton and membrane remodeling occurring at the midbody. These events are finely orchestrated by several , such as Aurora-B, , and , which act on multiple targets to regulate the local levels, the interactions, and the functions of key players of cytokinesis [3,7,8].

Cells 2019, 8, 684; doi:10.3390/cells8070684 www.mdpi.com/journal/cells Cells 2019, 8, 684 2 of 12

Spastin belongs to the family of the AAA (associated with diverse cellular activities) ATPases. The ATPase domain (342–599 aa) is preceded by the MT binding domain (MTBD, 270–328 aa) and the MT-interacting and endosomal-trafficking domain (MIT, 116–194 aa) that is necessary for midbody recruitment through CHMP1B interaction [4,5]. Four spastin isoforms have been identified, generated by two alternative start codons (full-length M1 and the shorter isoform lacking the first 86 aa, M87) and differential splicing of the exon 4, M1∆4, and M87∆4, respectively [6,9]. M87 is ubiquitously expressed, whereas M1 expression appears to be restricted to neuronal cells [6,9,10]. Spastin has been reported to localize at regions of active MT remodeling and its depletion results in strongly delayed abscission, associated with aberrant midbody phenotype [5,11,12]. In interphase, spastin shows a diffuse staining in the nucleus and a punctate pattern in the cytoplasm, where it localizes at the , endoplasmic reticulum, endosomes, and lipid droplets [13–15]. Instead, during cell division, spastin localizes at the spindle poles, at the resealing nuclear envelope, and at the midbody [5,15,16]. HIPK2 is a highly conserved multifunctional tyrosine-regulated serine/threonine kinase [17–19], which phosphorylates a large number of targets, contributing to the fine-tuning of several pathways [20,21]. We previously observed that HIPK2-null or -depleted cells undergo cytokinesis delay and failure, accumulating elongated midbodies and tetraploid cells [22,23]. During cytokinesis, HIPK2 is recruited at the midbody in an Aurora-B-dependent manner [24] and, there, phosphorylates the extrachromosomal histone H2B at S14 [22]. This phosphorylation is not required for the midbody localization of H2B, but it is necessary for successful cytokinesis [22,24], supporting the inclusion of HIPK2 among the kinases that regulate cytokinesis. Since these kinases usually work on multiple targets, we searched for additional cytokinesis substrates of HIPK2. Here, we identify spastin as a novel abscission target of HIPK2 and show that HIPK2-mediated phosphorylation of spastin at S268 is required for the midbody localization of spastin to drive abscission.

2. Materials and Methods

2.1. Conditions and Expression Vectors

HeLa, U2OS, and A549 cells were cultured at 37 ◦C and 5% CO2 in DMEM GlutaMAX, supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA); they were enriched in as in [22]. The following plasmids were employed: Flag-myc empty vector (pCMV6-Entry) and flag-myc-tagged spastin-expressing vectors from OriGene Technologies (Rockville, MD, USA). Spastin mutants were obtained by site-directed mutagenesis in the Spastin-flag-myc-tagged vector using a QuikChange Lightening Kit (Agilent, Cernusco sul Naviglio, Italy) and analyzed by sequencing. Vectors were transfected by using Lipofectamine LTX and Plus reagent (Life Technologies).

2.2. RNA Interference (RNAi) and Real-Time (Reverse Transcription)-PCR (Real-Time (RT)-PCR) HIPK2 RNAi was obtained by using human-specific validated stealth siRNAs (a mix of three different siRNAs by Life Technologies, as in [22]). Spastin RNAi was obtained by using a mix of specific validated stealth siRNAs by Life Technologies, targeting sequences common to all spastin isoforms. siRNAs were transfected using Lipofectamine RNAi MAX (Life Technologies). RNA extraction and real-time (RT)-PCR were performed as in [22], and relative fold-change were determined by the 2-∆∆Ct method using GAPDH mRNA as normalizer. Primers for amplification were as in [22]. SiRNA sequences were the following: siHIPK2#1: CCCGAGUCAGUAUCCAGCCCAAUUU; siHIPK2#2: CCACCAACCUGACCAUGACCUUUAA; siHIPK2#3: CAGGGUUUGCCUGCUGAAUAUUUAU; siSpastin#1: CCAGUGAGAUGAGAAAUAUUCGAUU; siSpastin#2: CCAGUGAGAUGAGAAAUAUUCGAUU. Cells 2019, 8, 684 3 of 12

2.3. Western Blot (WB) and Co-Immunoprecipitation (co-IP) For WB, total cell extracts (TCEs) were prepared in RIPA buffer (50mM Tris-HCl (pH 8), 600 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40 and 1mM EDTA) supplemented with protease and inhibitors (Roche, Basel, Switzerland). For co-IP, TCEs were prepared in non-denaturating lysis buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP40 and 5 mM EDTA), supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). Proteins were resolved by SDS-PAGE using Bolt Novex Bis-Tris Gels 4%–12% (Life Technologies). Immunoreactivity was determined using ECL-Prime (Amersham, Buckinghamshire, UK), image acquisition and densitometric analysis were performed with Image Lab software (BIORAD, Hercules, CA, USA). The following antibodies (Abs) were employed: anti-HIPK2 (rat monoclonal Ab C5C6, kindly provided by Dr. L. Schmitz); anti-GST (1:500), anti-GAPDH (1:1000), anti-His tag (1:200), and anti-spastin (1:100; sp311/1 mouse monoclonal Ab), all provided by Santa Cruz Biotechnology, Dallas, TX, USA; anti-alpha-tubulin (1:1000; Immunological Science, Rome, Italy); anti-Flag (mouse monoclonal Ab, TA50011, OriGene Technologies, and rabbit polyclonal Ab provided by Sigma-Aldrich, St. Louis, MO, USA); anti-GFP (mouse monoclonal Ab provided by Roche and rabbit polyclonal Ab provided by Santa Cruz Biotechnology); and anti-HRP-conjugated goat anti-mouse,-rat, and -rabbit (Cell Signaling Technology, Danvers, MA, USA).

2.4. Kinase Assay and Recombinant Proteins In vitro kinase assays were performed as in [19], by using HIPK2 Kinase domain (kind gift of Dr. Montemiglio) as the enzymatic source and the following recombinant proteins as substrates: His6-tagged spastin 79–310 (ag18866, Proteintech, Rosemont, IL, USA) and spastin M1 (TP320458, OriGene technologies).

2.5. GST Pull-Down and Binding Assay GST pull-down and binding assay were performed as in [24]. In particular, GST and GST-HIPK2 were obtained by H1299 T7-vaccinia virus infection followed by transfection as described in [22] and were incubated with 100 ng of recombinant His6-tagged spastin 79–310 (Proteintech) in 50 mM Tris-HCl, pH 7.5, 150 nM NaCl. Bound Spastin was analyzed by WB.

2.6. Mass Spectrometry (MS) In vitro kinase assays were performed with cold ATP, the products were resolved by NuPAGE 4%–12% (Life Technologies), and stained with the Colloidal Blue Staining kit (Life Technologies). The stained bands were cut from the gel and in gel digested with trypsin. The peptide mixture was analyzed by nanoflow-reversed-phase liquid chromatography tandem MS using an HPLC Ultimate 3000 (DIONEX, Sunnyvale, CA, USA) connected in line with a linear ion trap (LTQ, ThermoElectron, Waltham, MA, USA). MS/MS spectra were acquired in data-dependent mode, allowing the fragmentation of the five most intense ions detected in the full scan and an additional fragmentation when occurring a neutral loss of 49 or 32 Dalton.

2.7. p-S268 Spastin Ab Production and Purification Rabbit immunization was performed with the following amide-conjugated phosphopeptide SGHHRAP(pS)YSGLSMV. The obtained serum was positive/negative affinity, purified by using phosphorylated/non-phosphorylated peptides (Life Technologies).

2.8. Immunofluorescence (IF) and Quantitative Analysis of Signals Cells were seeded onto poly-L-lysine-coated coverslips, fixed in 2% formaldehyde or in ice-cold methanol, permeabilized in 0.25% Triton X-100 in PBS for 10 min, and then blocked in 5% bovine serum albumin in PBS before the primary Ab was applied. For each employed Ab and IF condition Cells 2019, 8, 684 4 of 12 see Supplementary Table S1. Appropriate secondary FITC- or TRITC-conjugated Abs (Alexa-fluor, Life Technologies) were used. DNA was marked with DAPI (Sigma-Aldrich). Preparations were examined under an Olympus AX70 microscope using a 100 /1.35 NA objective. Images for each × sample were taken in parallel using identical microscope settings. Signals were measured by using Image J software. Quantification of IF signals were performed as follows: (a) p-S268 spastin at the midbody: mean of pixel intensity at midbody corrected for external background; and (b) for MT density quantification, β-tubulin fluorescence intensity was measured by drawing cellular outline and measuring the mean of pixel intensity corrected for external background.

2.9. MT Depolymerization Assay Cells were placed on ice for indicated times in the presence of the MT depolymerizing agent 10 µM nocodazole (Sigma-Aldrich), fixed and then processed by IF. Hyperstable MTs were visualized by using anti-acetylated-tubulin.

2.10. Statistics Statistical analyses were performed using the GraphPad Prism software (GraphPad Software Inc., CA, USA). Data were tested for normality using Shapiro–Wilk normality test. The unpaired t-test or Chi-square (χ2test) was applied to determine the significance of quantitative experiments when the data distribution was normal. The Mann–Whitney test was used when the populations did not show a Gaussian distribution. Statistical significance was set at p < 0.05.

3. Results

3.1. Spastin is Not Detectable at the Midbody in HIPK2-Depleted Cells To identify new HIPK2 targets in cytokinesis, we examined the midbody localization of several structural and functional cytokinesis factors at the single-cell level by IF. Previous works have shown that acute HIPK2 depletion results in stronger cytokinesis defects when compared with chronic HIPK2 depletion, due to compensatory events [22,23]. Thus, we made our analyses upon acute HIPK2-depletion (siHIPK2) obtained by RNAi [22]. IF was performed when HIPK2 mRNA and protein levels were both strongly reduced when compared with control cells (siCtr) (Figure1A). We observed that HIPK2 depletion does not inhibit the midbody localization of cytokinesis master regulative kinases, such as Aurora B complex and PLK-1, proteins involved in the formation/stabilization of the midbody, such as Citron kinase, MKLP1, and ECT2, and abscission factors, such as Cep55, ALIX, and CHMP1B [25] (Figure1B,C). During this analysis, the only factor whose localization was strongly impaired by HIPK2 depletion was spastin, which was not detectable at the midbody in a high percentage of siHIPK2 cells (Figure1B,C). These results suggest that HIPK2 regulates spastin localization at the midbody. Cells 2019, 8, 684 5 of 12 Cells 2019, 8, 684 5 of 12

FigureFigure 1. 1.Spastin Spastin localization localization at at midbody midbody isis reducedreduced inin HIPK2-depletedHIPK2-depleted cells. cells. (A (,AC,)C HeLa) HeLa cells cells transfectedtransfected with with a mix a mix of of three three validated validated HIPK2-specific HIPK2-specific (siHIPK2) or or negative negative control control (siCtr) (siCtr) stealth stealth siRNAssiRNAs were were analyzed analyzed by by real-time real-time (RT)-PCR, (RT)-PCR, WBWB andand IFIF 96h96h postpost transfection. Molecular Molecular weight weight markersmarkers are reportedare reported here here and inand the in following the following figures figures in kilodalton in kilodalton (kDa). (kDa). Values Values are mean are meanstandard ± ± errorstandard of mean error (SEM) of mean from (SEM) two independent from two independen experiments,t experiments, each performed each performed in triplicate. in triplicate. **** p < ****0.0001,p < 0.0001, t-test. Representative WB is shown and relative densitometric values of HIPK2/GAPDH are t-test. Representative WB is shown and relative densitometric values of HIPK2/GAPDH are reported reported below each lane. IF was performed with Abs, recognizing indicated proteins in combination below each lane. IF was performed with Abs, recognizing indicated proteins in combination with with anti-α- or -β-tubulin, to mark midbody and DAPI to stain DNA. In (B), representative anti-α- or -β-tubulin, to mark midbody and DAPI to stain DNA. In (B), representative immunostainings immunostainings of midbody magnification are shown. Bar, 2 µM. In (C), data quantification is of midbody magnification are shown. Bar, 2 µM. In (C), data quantification is shown, values are mean shown, values are mean ± SEM from three independent experiments, each performed in duplicate, SEM from three independent experiments, each performed in duplicate, and >100 midbodies were ± and >100 midbodies were analyzed per condition. Note that, to avoid alterations of the IF signals, analyzedsiHIPK2 per midbodies condition. showing Note that, >2 tubulin-labelled to avoid alterations puncta of were the IF excluded signals, by siHIPK2 this analysis midbodies; ***p < showing 0.001 >2 tubulin-labelled(p = 0.0002), t-test. puncta were excluded by this analysis; *** p < 0.001 (p = 0.0002), t-test. 3.2. HIPK2-Depleted Cells Show Abscission Defects Similar to Spastin-Depleted Cells 3.2. HIPK2-Depleted Cells Show Abscission Defects Similar to Spastin-Depleted Cells Next, we evaluated whether siHIPK2 cells show functional defects compatible with the reduction Next, we evaluated whether siHIPK2 cells show functional defects compatible with the of spastin-positivereduction of spastin-positive midbody. Consistent midbody. with Consistent the spastin with role the in spastin MT severing role in andMT thesevering expectation and the that lossexpectation of spastin resultsthat loss in of greater spastin MT results stabilization in greater [MT26], stabilization we observed [26], hyperstability we observed of hyperstability the midbody of MT in siHIPK2the midbody cells MT when in siHIPK2 analyzed cells by when cold analyzed MT depolymerization by cold MT depolymerization assay in the presence assay in ofthe nocodazole presence (Figureof nocodazole2A,B). Furthermore, (Figure 2A,B). we Furt observedhermore, that we the observed midbodies that the of midbodies siHIPK2 cells of siHIPK2 resemble cells the resemble aberrant phenotypethe aberrant as previously phenotype described as previously in spastin-depleted described in spastin-depleted cells, i.e., MT-filled cells,elongated i.e., MT-filled midbodies elongated often associatedmidbodies with often tubulin-labelled associated with puncta tubulin-labelled [5] (Figure puncta2C–E). [ From5] (Figure here 2C–E). on, we From will here generally on, we refer willto thesegenerally phenotypes refer asto “aberrantthese phenotypes midbodies”. as “aberrant Comparable midbodies”. results Comparable were obtained results in HeLawere cellsobtained depleted in of bothHeLa HIPK2 cells depleted and spastin of both (data HIPK2 not shown) and spastin in other (data human not shown) HIPK2-depleted in other human cells (Figure HIPK2-depleted S1A) and by transfecting single HIPK2-specific siRNA, ruling out RNAi-off target effects (Figure S1B,C).

Cells 2019, 8, 684 6 of 12

cells (Figure S1A) and by transfecting single HIPK2-specific siRNA, ruling out RNAi-off target effects (Figure S1B–C). Cells 2019, These8, 684 results show a link between abscission defects and the reduction of spastin-positive6 of 12 midbodies in HIPK2-depleted cells, suggesting a role for HIPK2 upstream of spastin.

FigureFigure 2. HIPK2-depleted 2. HIPK2-depleted cells cells show show aberrant aberrant midbodies. midbodies. (A (,AB,)B Indicated) Indicated HeLa HeLa cells cells were were analyzed analyzed by IF 96by h IF post 96 transfectionh post transfection at the indicatedat the indicated time posttime nocodazolepost nocodazole (Noc) (Noc) treatment treatment on ice. on ice. Telophase Telophase cells, showingcells, acetylatedshowing tubulinacetylated at thetubulin midbody, at werethe midbody, scored. In (wereA), representative scored. In immunostainings(A), representative are shown.immunostainings Bar, 5 µM. In (areB), shown. the percentage Bar, 5 µM. of cellsIn (B with), the acetylatedpercentage tubulin of cells atwith the acetylated midbody tubulin are reported at the as meanmidbodySEM fromare reported two independent as mean experiments,± SEM from eachtwo independent performed in experiments, duplicate, in each which performed at least a totalin ± of 80duplicate, midbodies in which per condition at least a were total counted.of 80 midbodies (C–E) HeLaper condition cells were were transfected counted. ( asC– inE) FigureHeLa cells1A or were with spastin-specifictransfected as validated in Figure stealth 1A or siRNAswith spastin-specif and analyzedic validated 96 h post stealth transfection siRNAs by and WB analyzed (C) and by96 IFh post (D,E ). In (Ctransfection), the arrow by indicates WB (C) theand band by IF corresponding (D,E). In (C), the to M87, arrow the indicates most abundant the band spastin corresponding form; M87 to ∆M87,4 was generallythe most less abundant abundant spastin and barely form; detectable.M87∆4 was Ingenerally (D), representative less abundant immunostainings and barely detectable. of siHIP2 In (D and), siSpastinrepresentative telophase immunostainings cells with aberrant of midbodysiHIP2 and are siSp shown.astin telophase The arrows cells indicate with aberrant tubulin-labeled midbody puncta are alongshown. the intercellular The arrows bridge.indicate Bar, tubulin-labeled 5 µM. In (E ),puncta the percentage along the ofintercellular cells showing bridge. aberrant Bar, 5 midbodiesµM. In (E), is reportedthe percentage as mean ofSEM cells from showing three independentaberrant midbodies experiments, is reported in which as atmean least ± total SEM 150 from midbodies three independent experiments,± in which at least total 150 midbodies per condition were analyzed. ****p < per condition were analyzed. **** p < 0.0001, χ2 test. 0.0001, χ2 test. These results show a link between abscission defects and the reduction of spastin-positive 3.3. HIPK2 Phosphorylates Spastin at S268 midbodies in HIPK2-depleted cells, suggesting a role for HIPK2 upstream of spastin. Thus, we evaluated whether spastin is a direct target of HIPK2. By co-IP experiments and in 3.3.vitro HIPK2 assays, Phosphorylates we showed Spastin that at S268HIPK2 binds and phosphorylates spastin (Figure 3). We co- immunoprecipitedThus, we evaluated epitope-tagged whether spastin flag-myc-spastin is a direct target and ofGFP–HIPK2, HIPK2. By by co-IP transiently experiments expressing and inthese vitro proteins in HeLa cells and immunoprecipitating with anti-Flag or anti-GFP Abs (Figure 3A). In in assays, we showed that HIPK2 binds and phosphorylates spastin (Figure3). We co-immunoprecipited vitro assays, HIPK2 was able to bind and phosphorylate a recombinant spastin fusion protein, i.e., a epitope-tagged flag-myc-spastin and GFP–HIPK2, by transiently expressing these proteins in HeLa His6-tagged fragment spanning aa 79–310 produced in bacteria (Figure 3B,C). In addition, HIPK2 was cells and immunoprecipitating with anti-Flag or anti-GFP Abs (Figure3A). In in vitro assays, HIPK2 able to phosphorylate the flag-myc-spastin full-length M1 produced in mammalian cells (Figure 3D). was able to bind and phosphorylate a recombinant spastin fusion protein, i.e., a His6-tagged fragment spanning aa 79–310 produced in bacteria (Figure3B,C). In addition, HIPK2 was able to phosphorylate the flag-myc-spastin full-length M1 produced in mammalian cells (Figure3D). Furthermore, by MS analysis, performed on the recombinant His6-tagged spastin in vitro phosphorylated by HIPK2, we identified spastin-S268 as a HIPK2 phosphorylation site (Figure3E). Cells 2019, 8, 684 7 of 12

Furthermore, by MS analysis, performed on the recombinant His6-tagged spastin in vitro phosphorylated by HIPK2, we identified spastin-S268 as a HIPK2 phosphorylation site (Figure 3E). S268 is an evolutionary conserved residue (Figure S2A), common to all spastin isoforms, located in a disordered region between the MIT and the MTBD domain. This residue has been reported to be phosphorylated in vivo by large-scale MS analysis performed in human and mouse cells (https://www.phosphosite.org). In addition, it is preceded by a proline, a consensus motif already reported for other HIPK2 targets [27]. Thus, we generated vectors expressing non-phosphorylatable or phosphomimetic spastin derivatives (S268A and S268D, respectively), and produced and validated an Ab that specifically recognizes spastin phosphorylated at S268 (p-S268 Ab; Figure S2B,C). Cells 2019, 8, 684 7 of 12

Figure 3. HIPK2 phosphorylates spastin at S268. (A) HeLa cells were transfected with the indicatedFigure 3. combination HIPK2 phosphorylates of vectors. spastin Exogenous at S268. flag-myc-tagged (A) HeLa cells spastin,were transfected expressing with both the M1indicated and M87 isoforms,combinationwas immunoprecipitated of vectors. Exogenouswith flag-myc-tagged anti-Flag (mouse spastin, Ab expressing by OriGene both Technologies), M1 and M87or isoforms, exogenous GFP-taggedwas immunoprecipitated HIPK2 was immunoprecipitated with anti-Flag (mouse with Ab anti-GFP by OriGene (mouse Technologies), Ab by Roche). or exogenous WB for indicated GFP- proteinstaggedwas HIPK2 performed was immunoprecipitated by using anti-Flag with (rabbit anti-GFP Ab by (mouse Sigma-Aldrich) Ab by Roche). and anti-GFPWB for indicated (rabbit Ab byproteins Santa Cruz was performed Technology). by using Diff anti-Flagerent exposure (rabbit Ab times by Sigma-Aldrich) for TCE and IPand are anti-GFP shown. (rabbit (B) GSTAb by and GST-HIPK2Santa Cruz proteins Technology). were producedDifferent exposu by T7-vacciniare times for system TCE and in H1299IP are shown. cells, purified (B) GST byand GST GST-HIPK2 pull-down, proteins were produced by T7-vaccinia system in H1299 cells, purified by GST pull-down, and and incubated with an equal amount of recombinant His6-tagged spastin 79–310. Spastin binding incubated with an equal amount of recombinant His6-tagged spastin 79–310. Spastin binding was was detected by WB. Input = 50 ng of recombinant spastin. Representative WB is shown (blot was detected by WB. Input = 50 ng of recombinant spastin. Representative WB is shown (blot was vertically cropped to eliminate non-related samples). (C) In vitro kinase assay was performed by using vertically cropped to eliminate non-related samples). (C) In vitro kinase assay was performed by HIPK2 kinase domain (HIPK2) as the enzymatic source and His6-tagged spastin 79–310 as the substrate using HIPK2 kinase domain (HIPK2) as the enzymatic source and His6-tagged spastin 79–310 as the in presence of γ-ATP-P32. Representative autoradiography and relative Coomassie blue staining are substrate in presence of γ-ATP-P32. Representative autoradiography and relative Coomassie blue shown. (D) In vitro kinase assay, performed as in C by using flag-myc-tagged spastin full-length M1 staining are shown. (D) In vitro kinase assay, performed as in C by using flag-myc-tagged spastin asfull-length the substrate. M1 as Representative the substrate. Representative autoradiography autoradiography and relative and Coomassie relative Coomassie blue staining blue arestaining shown. (D)areIn shown. vitro kinase (D) Inassay, vitro kinase performed assay, asperformed in C, by as using in C, cold by using ATP cold and ATP analyzed and analyzed by MS. by The MS. ion The 731.9, correspondingion 731.9, corresponding to the phosphorylated to the phosphorylated spastin peptide spastin (266–279) peptide encompassing(266–279) encompassing S268, was S268, fragmented, was producingfragmented, a MS2 producing spectrum a MS2 with spectrum the most intensewith the ion most 628.9. intense This ion ion 628.9. revealed This aion 49 revealed Da neutral a 49 loss Da and 3 wasneutral further loss fragmented and was further in the fragmented MS spectrum in the that MS is3 spectrum shown. that is shown.

S268 is an evolutionary conserved residue (Figure S2A), common to all spastin isoforms, located in a disordered region between the MIT and the MTBD domain. This residue has been reported to be phosphorylated in vivo by large-scale MS analysis performed in human and mouse cells (https://www.phosphosite.org). In addition, it is preceded by a proline, a consensus motif already reported for other HIPK2 targets [27]. Thus, we generated vectors expressing non-phosphorylatable or phosphomimetic spastin derivatives (S268A and S268D, respectively), and produced and validated an Ab that specifically recognizes spastin phosphorylated at S268 (p-S268 Ab; Figure S2B,C). Cells 2019, 8, 684 8 of 12

3.4. Spastin-S268 Phosphorylation is Present at the Midbody and is Required for Abscission To evaluate whether the midbody localized spastin is phosphorylated at S268, we performed IF with p-S268 Ab. A clear immunostaining was detected at the midbody in the control cells (Figure 4A,B; siCtr panels), while a significant decrease of this staining was observed in siHIPK2 and siSpastin cells, both without spastin at the midbody, confirming the absence of non-specific Ab signals in IF (Figure 4A,B). To verify the contribution of HIPK2 in spastin-S268 phosphorylation, we restored HIPK2 expression in the siHIPK2 cells by transfection of siRNA-resistant HIPK2 forms. Expression of the HIPK2-WT form reestablished spastin-S268 phosphorylation at the midbody. In contrast, the kinase- defective form, i.e., the HIPK2-K228R mutant, did not restore spastin phosphorylation (Figure 4C), indicating that the HIPK2 kinase activity is required for spastin-S268 phosphorylation at the midbody. Next, we evaluated whether this phosphorylation contributes to the cytokinetic localization of spastin by assessing the capability of the non-phosphorylatable spastin-S268A mutant to localize at the midbody. We found that, in contrast with the phosphomimetic S268D forms, spastin-S268A was barely detectable at the midbody (Figure 5A). As expected, the midbody localization of these spastin mutants was independent of HIPK2 (Figure 5A, right columns). Then, we asked whether expression Cells 8 of2019 the, phosphomimetic, 684 spastin-S268D mutant could rescue the abscission defects of siHIPK2 cells.8 of 12 Microscopy assessment of aberrant midbodies and binucleated cells showed a significant reduction 3.4.in Spastin-S268 the percentage Phosphorylation of defects, following Is Present expression at the Midbody of S268D and (Figure Is Required 5B–D). for A Abscission comparable effect was obtained following expression of spastin-WT, even if spastin-S268D resulted more efficient than -WT in Tothese evaluate analyses whether (Figure the 5B–D). midbody In contrast, localized the spastinnon-phosphorylatable is phosphorylated S268A at mutant, S268, we according performed to IF withits p-S268impaired Ab. midbody A clear immunostaininglocalization, was wasunable detected to rescue at theabscission midbody in insiHIPK2 the control cells cells(Figure (Figure 5B–D),4A,B; siCtralthough panels), it maintained while a significant its enzymati decreasec MT-severing of this activity staining (Figure was observed S3A). in siHIPK2 and siSpastin cells, bothThese without results spastinindicate at that the HIPK2-mediated midbody, confirming S268 phosphorylation the absence of of non-specific spastin contributes Ab signals to its in IF (Figuremidbody4A,B). localization and activity for abscission.

FigureFigure 4. 4.Analysis Analysis of of spastin-S268 spastin-S268 phosphorylationphosphorylation at at the the midbody. midbody. (A ()A Indicated) Indicated HeLa HeLa cells cells were were fixedfixed and and stained stained with with DAPI, DAPI, anti- anti-ββ-tubulin,-tubulin, and and p-S268 p-S268 Abs,Abs, andand analyzedanalyzed to quantify quantify the the IF IF signal signal of p-S268of p-S268 spastin spastin at the at midbody. the midbody. Representative Representative immunostainings immunostainings of midbody of midbody magnification magnification are shown.are Bar,shown. 1 µM. Bar,Quantification 1 μM. Quantification in arbitrary in arbitrary units (a.u.) units is reported(a.u.) is reported in the dot in plotthe dot showing plot showing the single the measures,single asmeasures, well as, the as meanwell as, value the meanSEM value from ± SEM two from different two experimentsdifferent experiments **** p < ****0.0001,p < 0.0001, Mann–Whitney Mann– ± test.Whitney (B) Indicated test. (B HeLa) Indicated cells were HeLa stained cells andwere analyzed stained asand in A.analyzed Representative as in A. immunostainings Representative of midbodyimmunostainings magnification of midbody are shown. magnification Bar, 1 µM. areQuantification shown. Bar, 1 isµM. reported Quantification in the dot is reported plot showing in the the singledot measures,plot showing as wellthe single as, the measures, mean value as wellSEM as,from the mean three divaluefferent ± SEM experiments from three **** differentp < 0.0001, ± Mann–Whitneyexperiments **** test.p < (0.0001,C) Dot Mann–Whitney plot showing the test. quantification (C) Dot plot showing of the p-S268 the quantification spastin IF signal of the in p-S268 indicated spastin IF signal in indicated HeLa cells 24 h upon transfection with low doses of vectors expressing HeLa cells 24 h upon transfection with low doses of vectors expressing GFP-tagged murine HIPK2-WT GFP-tagged murine HIPK2-WT or its kinase defective derivative. siHIPK2 cells were transfected with or its kinase defective derivative. siHIPK2 cells were transfected with indicated vectors 48 h post siRNA indicated vectors 48 h post siRNA transfection performed as in Figure 1A and analyzed 48 h post transfection performed as in Figure1A and analyzed 48 h post vector transfection by IF. Note that, vector transfection by IF. Note that, in siHIPK2 cells, the expression of murine HIPK2 protein, in siHIPK2 cells, the expression of murine HIPK2 protein, showing >97% identity with human HIPK2 showing >97% identity with human HIPK2 and fully recapitulating its functions, is not inhibited by and fully recapitulating its functions, is not inhibited by RNAi because human-specific siRNAs were RNAi because human-specific siRNAs were used. ***p < 0.001(p = 0.0005) t-test. used. *** p < 0.001(p = 0.0005) t-test.

To verify the contribution of HIPK2 in spastin-S268 phosphorylation, we restored HIPK2 expression in the siHIPK2 cells by transfection of siRNA-resistant HIPK2 forms. Expression of the HIPK2-WT form reestablished spastin-S268 phosphorylation at the midbody. In contrast, the kinase-defective form, i.e., the HIPK2-K228R mutant, did not restore spastin phosphorylation (Figure4C), indicating that the HIPK2 kinase activity is required for spastin-S268 phosphorylation at the midbody. Next, we evaluated whether this phosphorylation contributes to the cytokinetic localization of spastin by assessing the capability of the non-phosphorylatable spastin-S268A mutant to localize at the midbody. We found that, in contrast with the phosphomimetic S268D forms, spastin-S268A was barely detectable at the midbody (Figure5A). As expected, the midbody localization of these spastin mutants was independent of HIPK2 (Figure5A, right columns). Then, we asked whether expression of the phosphomimetic spastin-S268D mutant could rescue the abscission defects of siHIPK2 cells. Microscopy assessment of aberrant midbodies and binucleated cells showed a significant reduction in the percentage of defects, following expression of S268D (Figure5B–D). A comparable e ffect was obtained following expression of spastin-WT, even if spastin-S268D resulted more efficient than -WT in these analyses (Figure5B–D). In contrast, the non-phosphorylatable S268A mutant, according to its impaired midbody localization, was unable to rescue abscission in siHIPK2 cells (Figure5B–D), although it maintained its enzymatic MT-severing activity (Figure S3A). CellsCells2019 2019, 8, 684, 8, 684 9 of 912 of 12

Figure 5. Spastin-S268D restores abscission in siHIPK2 cells. (A) HeLa control cells and siHIPK2 cells Figure 5. Spastin-S268D restores abscission in siHIPK2 cells. (A) HeLa control cells and siHIPK2 cells were transfected with vectors expressing flag-myc-tagged spastin-S268A or -S268D and analyzed 48 h were transfected with vectors expressing flag-myc-tagged spastin-S268A or -S268D and analyzed 48 post transfection by IF after staining with DAPI, anti-β-tubulin, and anti-flag Ab to visualize midbody h post transfection by IF after staining with DAPI, anti-β-tubulin, and anti-flag Ab to visualize localizationmidbody oflocalization exogenous of spastin. exogenous Representative spastin. Representative immunostainings immunostainings of midbody magnification of midbody are shown in the left panels. Bar, 1 µM. Data quantification are reported, the values are mean SEM from magnification are shown in the left panels. Bar,1 µM. Data quantification are reported, the values± are threemean independent ± SEM from experiments. three independent Similar experiments. data were obtainedSimilar data by usingwere obtained anti-myc by Ab using to stain anti-myc exogenous Ab spastinto stain (data exogenous not shown). spastin (B–D (data) siHIPK2 not shown). cells were (B– transfectedD) siHIPK2 with cells flag-myc were transfected empty vector with orflag-myc indicated flag-mycempty spastin-expressingvector or indicated vectors flag-myc 48 hspastin-expr post siRNAessing transfection vectors 48 performed h post siRNA as in Figuretransfection1A and analyzedperformed 48 h as post in Figure vector transfection1A and analyzed by WB 48 andh post IF aftervector staining transfection with by anti-Flag, WB and anti- IF afterβ-tubulin, staining and DAPI.with In anti-Flag, (B), representative anti-β-tubulin, WB and are DAPI. shown. In (B In), representative (C), the percentage WB are of shown. Flag-positive In (C), the cells, percentage showing aberrantof Flag-positive midbodies, cells, is reported showing as mean aberrantSEM midbod from threeies, is independent reported as experiments, mean ± SEM each from performed three in ± duplicate,independent in which experiments, at least a each total performed of 80 midbodies in duplicat per conditione, in which were at least analyzed. a total of In 80 (D midbodies), the percentage per of Flag-positivecondition were binucleated analyzed. In cells(D), the is reportedpercentage as of fold-change Flag-positiverelative binucleated to that cells ofis reported vector-transfected as fold- cellschange in two relative independent to that of experiments, vector-transfected in which cells total in two>2000 independent cells were experiments, scored. The in percentages which total of Flag-positive>2000 cellsbinucleated were scored. cells The (mean percentagesSEM) of are Flag-positive the following: binucleated 4 0.5, 1.95 cells 0.55,(mean 3 ± 0.30,SEM) 1.25are the0.25 ± ± ± ± ± forfollowing: siHIPK2 cells 4 ± 0.5, expressing 1.95 ± 0.55, empty 3 ± 0.30, Vector, 1.25 spastin-WT, ± 0.25 for siHIPK2 -S268D, cells and expressing -S268A, respectively. empty Vector, **** spastin-p < 0.0001, χ2 *** pWT,< 0.001( -S268D,p = and0.0002) -S268A,χ2 test.respectively. ****p < 0.0001, ***p < 0.001(p = 0.0002) test.

4. TheseDiscussion results indicate that HIPK2-mediated S268 phosphorylation of spastin contributes to its midbodyIn localizationthis study, we and showed activity that for abscission.HIPK2 contributes to abscission through phosphorylation of spastin at S268. We observed the presence of phospho-S268 spastin at the midbody and a strong 4. Discussionreduction of this spastin localization in HIPK2-depleted cells, associated with cytokinesis defects, whichIn this can study, be rescued we showed by expressing that HIPK2 phosphomimetic contributes to spastin abscission mutant through (i.e., S268D), phosphorylation supporting of a spastinkey at S268.role of We HIPK2-mediated observed the presence spastin of phosphorylation phospho-S268 spastin for spastin at the midbodymidbody and localization a strong reductionto allow offor this spastinsuccessful localization abscission. in HIPK2-depleted cells, associated with cytokinesis defects, which can be rescued by expressingThus far, phosphomimetic several studies have spastin been mutant carried (i.e., out to S268D), investigate supporting the mechanisms a key role involved of HIPK2-mediated in spastin regulation. It has been demonstrated that spastin is regulated by NRF1 and SOX11 transcription spastin phosphorylation for spastin midbody localization to allow for successful abscission. factors, by miR-96, miR-182 and Elk1 [28,29]; however, spastin post-translational regulation Thus far, several studies have been carried out to investigate the mechanisms involved in spastin mechanisms are largely unknown. Notably, we identified a novel regulatory mechanism in which regulation. It has been demonstrated that spastin is regulated by NRF1 and SOX11 transcription factors, HIPK2 assures spastin midbody presence by phosphorylating it at S268. By MS and phospho-specific by miR-96,Ab we identified miR-182 HIPK2 and Elk1 as the [28 ,first29]; kinase however, that spastin phosphorylates post-translational spastin. Even regulation if our analysis mechanisms did not are largelypreclude unknown. the existence Notably, of weother identified HIPK2 aphosphorylation novel regulatory site(s), mechanism S268 phosphorylation in which HIPK2 appears assures to spastin be midbody presence by phosphorylating it at S268. By MS and phospho-specific Ab we identified HIPK2 as the first kinase that phosphorylates spastin. Even if our analysis did not preclude the existence Cells 2019, 8, 684 10 of 12

Cells 2019, 8, 684 10 of 12 of other HIPK2 phosphorylation site(s), S268 phosphorylation appears to be crucial for spastin in cytokinesis,crucial for as spastin we functionally in cytokinesis, showed as by we phosphomimetic functionally showed and non-phosphorylatable by phosphomimetic mutants.and non- phosphorylatableAt this point, how mutants. S268 phosphorylation regulates spastin localization during abscission remains to be clarified.At this Ourpoint, single-cell how S268 screening phosphorylation shows that regu HIPK2-depletionlates spastin impairslocalization the midbodyduring abscission localization of spastinremains without to be clarified. inhibiting Our thatsingle-cell of itsrecruiter screening CHMP1B shows that and HIPK2-depletion of other upstream impairs cytokinesis the midbody factors. Welocalization can hypothesize of spastin that S268without phosphorylation inhibiting that promotes of its recruiter midbody CHMP1B recruitment and byof stabilizingother upstream spastin interactioncytokinesis with factors. its recruiter We can or hypothesize other proteins, that or S268 induces phosphorylation conformational promotes changes midbody that aff recruitmentect its stability in thisby stabilizing subcompartment, spastin byinteraction ensuring with the concentration its recruiter or of thisother enzyme proteins, at theor endinduces of abscission, conformational when its MT-severingchanges that function affect its is stability necessary. in this By WBsubcompartment, analysis performed by ensuring on TCEs the concentration from telophase-enriched of this enzyme cells, at the end of abscission, when its MT-severing function is necessary. By WB analysis performed on we observed a reduction of spastin levels in the HIPK2-depleted cells when compared to their control TCEs from telophase-enriched cells, we observed a reduction of spastin levels in the HIPK2-depleted cells (Figure S3B), indicating that HIPK2 depletion reduces spastin levels, and not only its localization, cells when compared to their control cells (Figure S3B), indicating that HIPK2 depletion reduces at the midbody. Furthermore, we obtained similar data on TCE from asynchronous cells (Figure S3C), spastin levels, and not only its localization, at the midbody. Furthermore, we obtained similar data andon we TCE noticed from asynchronous a general decrease cells (Figure of spastin S3C), signals and we by noticed IF in a the general HIPK2-depleted decrease of spastin interphase signals cells (Figureby IF S3D), in the suggesting HIPK2-depleted that the interphase HIPK2-mediated cells (Figure regulation S3D), suggesting of spastin that might the notHIPK2-mediated be restricted to cytokinesis.regulation Interestingly,of spastin might katanin, not be a restricted microtubule-severing to cytokinesis. enzyme Interestingly, belonging katanin, to thea microtubule- same ATPase familysevering of spastin, enzyme is belonging regulated to via the phosphorylation same ATPase family in a of disordered spastin, is proteinregulated region via phosphorylation similar to that in whichin a S268disordered is located protein in spastinregion similar [30,31 ],to openingthat in which thepossibility S268 is located for ain more spastin general [30,31] mechanism, opening the for regulationpossibility of AAAfor a ATPases,more general presenting mechanism the same for regulation domain arrangement of AAA ATPases, as spastin. presenting Furthermore, the same both HIPK2domain and arrangement spastin have as been spastin. shown Furthermore, to play roles both in HIPK2 different and biological spastin have processes. been shown Our to data play open roles the possibilityin different that biological HIPK2 can processes. also function Our data as a open spastin the regulator possibility in that other HIPK2 spastin-dependent can also function processes, as a suchspastin as neurite regulator branching, in other axonal spastin-dependent growth, endosome processes, tubulation, such as neurite and nuclear branching, envelope axonal sealing growth, [32 ]. [ ] endosomePreviously, tubulation, we have and demonstrated nuclear envelope that the sealing expression 32 . of the phosphomimetic mutant H2B-S14D can restorePreviously, successful we have cytokinesis demonstrated in HIPK2-depleted that the expression cells of [ 22the,23 phosphomimetic]. We observed mutant that the H2B-S14D abscission [ ] defectscan restore were successful rescued incytokinesis siHIPK2 in cellsHIPK2-depleted expressing cells H2B-S14D 22,23 . We without observed aff ectingthat theS268 abscission spastin defects were rescued in siHIPK2 cells expressing H2B-S14D without affecting S268 spastin phosphorylation at the midbody (pS268 intensity at the midbody was 12.9 8.0 and 11.7 5.3 phosphorylation at the midbody (pS268 intensity at the midbody was 12.9 ± 8.0± and 11.7 ± 5.3 ±in in siHIPK2 and in siHIPK2+H2B-S14D cells, respectively). Similarly, these defects were rescued siHIPK2 and in siHIPK2+H2B-S14D cells, respectively). Similarly, these defects were rescued in in siHIPK2 cells expressing spastin-S268D, independently of H2B-S14 phosphorylation (pH2B-S14 siHIPK2 cells expressing spastin-S268D, independently of H2B-S14 phosphorylation (pH2B-S14 intensity at the midbody was 5.1 2.9 and 4.9 2.7 in siHIPK2 and in siHIPK2 cells+spastin-S268D, intensity at the midbody was 5.1± ± 2.9 and 4.9 ±± 2.7 in siHIPK2 and in siHIPK2 cells+spastin-S268D, respectively).respectively). These These observations observations suggest suggest twotwo parallelparallel mechanisms of of action action of of HIPK2 HIPK2 in incytokinesis cytokinesis (Figure(Figure6). 6). Further Further supporting supporting this this idea, idea, depletion depletion ofof extrachromosomalextrachromosomal H2B H2B had had no no effect e ffect on onthe the presencepresence of spastinof spastin at at the the midbody midbody (LM (LM and and SS,SS, manuscriptmanuscript in in preparation). preparation). Thus, Thus, similar similar to toother other cytokinesiscytokinesis regulating regulating kinases kinases [33 [33,34,34],], HIPK2 HIPK2 contributescontributes to to cytokinesis cytokinesis by by acting acting on on multiple multiple targets targets on dionff differenterent pathways. pathways.

FigureFigure 6. 6.SchematicSchematic representation representation of HIPK2 of HIPK2 activity activity on spastin on spastin and H2B and during H2B during cytokinesis. cytokinesis.

Author Contributions: C.R. designed the study, supervised the research activity, and wrote the manuscript. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/8/7/684/s1, FigureA.P., S1 L.B.,. Aberrant M.F., D.V., midbodies and F.S afterperformed HIPK2 most depletion; of the experiments.Figure S2. pS268 Kinase Ab assays validation; were performedFigure S3. bySpastin-S268A L.M.; D.V. MTand severing F.S. performed activity and formal spastin data levelsanalysis. in MS HIPK2-depleted was performed cells. by S.CTableS1: and M.C.List C.R. of and Abs S.S. and analyzed IF conditions. all data and edited/reviewed the manuscript. Author Contributions: C.R. designed the study, supervised the research activity, and wrote the manuscript. A.P., L.B.,Funding: M.F., D.V., This and work F.S. was performed supported most by grants of the from experiments. Fondazione Kinase Telethon assays (GGP16072), were performed AFM Telethon by L.M.; (#22157), D.V. and F.S.and performed Latium Region formal (85-2017-15348) data analysis. to MS C.R., was by performed Italian Association by S.C.and for Cancer M.C. C.R. Research and S.S. to C.R. analyzed (IG #17739) all data and and edited/reviewed the manuscript.

Cells 2019, 8, 684 11 of 12

Funding: This work was supported by grants from Fondazione Telethon (GGP16072), AFM Telethon (#22157), and Latium Region (85-2017-15348) to C.R., by Italian Association for Cancer Research to C.R. (IG #17739) and S.S. (IG #14592). L.M. and D.V. were recipients of fellowships from the Italian Foundation for Cancer Research. We are grateful to all people cited in the text for their gifts of cells and reagents. Acknowledgments: We thank F. Magi, B. Ciani, F. Degrassi, and E. Cundari for their help, M. Di Segni for the proofreading of the manuscript, and K. Bejio for technical assistance. Conflicts of Interest: The authors declare that they have no conflict of interest.

References

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