2274 Research Article

IntroductionMammalian NIMA (never in mitosis A)-related kinases (Nek1-Nek11) are a family of Ser-Thr kinases that constitute approximately2% of the entire human kinome (Fry and Nigg, 1995; O’Connellet al., 2003; O’Regan et al., 2007; Quarmby and Mahjoub, 2005;Schultz et al., 1993). They share approximately 40-45% identitywithin their N-terminal catalytic kinase domains, but the C-terminalnon-catalytic regions are highly divergent (Fry and Nigg, 1995; Fryet al., 1997). The founding member of the Nek family, NIMA, isessential for entry into mitosis in Aspergillus nidulans (O’Connellet al., 2003). Other homologues of NIMA, such as Fin1 and Kin3from yeast, also have important cell cycle functions via regulationof microtubule organization and mitotic spindle assembly (Jonesand Rosamond, 1990; Krien et al., 1998). In addition, theChlamydomonas NIMA-family kinases Fa2p and Cnk2p regulateciliary function and length by promoting cilia disassembly (Bradleyand Quarmby, 2005; Bradley et al., 2004; Finst et al., 2000; Finstet al., 1998; Mahjoub et al., 2002; Mahjoub et al., 2004; Parker etal., 2007; Quarmby and Mahjoub, 2005), and Nek from Crithidiadisplays tubulin polyglutamylation activity (Westermann and Weber,2002; Westermann and Weber, 2003). Finally, Nek kinases influenceorgan development and morphogenesis in Arabidopsis through theireffects on microtubules (Cloutier et al., 2005; Motose et al., 2008;Sakai et al., 2008; Vigneault et al., 2007). Thus, a common themein Nek function is the regulation of microtubule structures.

The functions of mammalian Nek proteins are largely unknown;however, similarly to their ancestral homologues, they are associatedwith cilia, centrosomes and microtubule organization (O’Regan etal., 2007; Quarmby and Mahjoub, 2005). For example, Nek2 iscrucial for proper mitosis through its effects on centrosomes (Fry

et al., 1998; Prigent et al., 2005), and Nek1 and Nek8 are involvedin primary cilium formation and polycystic kidney diseases(Mahjoub et al., 2005; Shalom et al., 2008; Upadhya et al., 2000).Nek9 phosphorylates Bicd2, a protein that is important formicrotubule maintenance and axonal stability (Holland et al., 2002;Teuling et al., 2008). Nek9 also modulates mitotic progression (Roiget al., 2002) and can activate Nek6 and Nek7 (Belham et al., 2003),which act on centrosomes and modulate microtubule nucleation(Kim et al., 2007; Yin et al., 2003). Nek6 can phosphorylate thekinesin Eg5, which regulates microtubule movement and isnecessary for spindle bipolarity during mitosis (Rapley et al., 2008).The involvement of Nek kinases in cell cycle control in particularhas stimulated intense scrutiny of their roles in cancer (Hernandezand Almeida, 2006; Kokuryo et al., 2007; Malumbres and Barbacid,2007; McHale et al., 2008; Miller et al., 2007; Miller et al., 2005;Simpson et al., 2008).

Although Nek kinases are most closely associated with cell cycleregulation and ciliogenesis, recent reports of high Nek expressionin the nervous system, including post-mitotic neurons, indicate thatthey might participate in other cellular processes (Arama et al., 1998;Feige and Motro, 2002; Tanaka and Nigg, 1999). For example, Nek3levels are higher in quiescent cells, and inhibition of Nek3 activityvia antibody injection or mutation did not affect cell cycleprogression (Tanaka and Nigg, 1999). Furthermore, although mostNek kinases are located in the centrosome or in cilia, Nek3 is foundin the cytoplasm (Tanaka and Nigg, 1999). Nek3 bears a conservedNIMA-related kinase domain at its N-terminus, and the C-terminalnon-catalytic region contains two PEST (proline-, glutamate-,serine- and threonine-rich) domains, a protein motif that isimplicated in promoting protein turnover or degradation as well as

NIMA-related kinases (Neks) belong to a large family of Ser/Thrkinases that have critical roles in coordinating microtubuledynamics during ciliogenesis and mitotic progression. The Nekkinases are also expressed in neurons, whose axonal projectionsare, similarly to cilia, microtubule-abundant structures thatextend from the cell body. We therefore investigated whetherNek kinases have additional, non-mitotic roles in neurons. Wefound that Nek3 influences neuronal morphogenesis andpolarity through effects on microtubules. Nek3 is expressed inthe cytoplasm and axons of neurons and is phosphorylated atThr475 located in the C-terminal PEST domain, which regulatesits catalytic activity. Although exogenous expression of wild-typeor phosphomimic (T475D) Nek3 in cultured neurons has nodiscernible impact, expression of a phospho-defective mutant

(T475A) or PEST-truncated Nek3 leads to distorted neuronalmorphology with disturbed polarity and deacetylation ofmicrotubules via HDAC6 in its kinase-dependent manner.Thus, the phosphorylation at Thr475 serves as a regulatoryswitch that alters Nek3 function. The deacetylation ofmicrotubules in neurons by unphosphorylated Nek3 raises thepossibility that it could have a role in disorders where axonaldegeneration is an important component.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/13/2274/DC1

Key words: Nek3, NIMA, Microtubule, Cilia, Axon, Neuron,Acetylation, BBS, HDAC6

Summary

The NIMA-family kinase Nek3 regulates microtubuleacetylation in neuronsJufang Chang1, Robert H. Baloh2,3 and Jeffrey Milbrandt1,3,*1Department of Pathology and Immunology, 2Department of Neurology and 3HOPE Center for Neurological Disorders, Washington UniversitySchool of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA*Author for correspondence (e-mail: jmilbrandt@wustl.edu)

Accepted 1 April 2009Journal of Cell Science 122, 2274-2282 Published by The Company of Biologists 2009doi:10.1242/jcs.048975

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protein-protein interactions (Pongubala et al., 1992; Rogers et al.,1986). Interestingly, a screen for phosphorylated proteins indeveloping mouse brain identified Nek3 (Ballif et al., 2004). Thephosphorylated residue was identified as Thr475, which is locatedwithin the C-terminal PEST domain. This residue and the PESTdomains are phylogenetically conserved, suggesting that they areimportant for Nek3 function.

In the present study, we found that several Nek kinases, includingNek3, are highly expressed in neurons from the central andperipheral nervous systems. Given the role of Nek kinases inmicrotubule regulation, we investigated whether Nek3 regulatesmicrotubules and therefore cytoskeletal dynamics in neurons. Wefound that cells expressing Nek3 mutants lacking either theconserved PEST domain or containing mutations of Thr475 hadseverely decreased levels of acetylated α-tubulin. Experiments withdeacetylase inhibitors showed that Nek3-mediated alterations intubulin acetylation resulted from HDAC6 modulation. Neuronsexpressing these mutants had abnormal morphology and multipleaxons, indicating disrupted cell polarity. These results suggest thatphosphorylation of Nek3 at T475 is an important regulator of Nek3activity. The regulation of neuronal microtubule acetylation by Nek3raises the possibility that it could be involved in disorders whereaxonal degeneration is an important component.

ResultsNek3 is expressed in post-mitotic neuronsNIMA-related kinases are crucial for regulating microtubulesduring mitosis; however, several Nek kinase mRNAs are presentin distinct neuronal structures in mouse brain and spinal cord (Aramaet al., 1998; Feige and Motro, 2002; Tanaka and Nigg, 1999). Asmicrotubule dynamics are important for the formation of neuronalprojections, these findings suggested that Nek kinases mightregulate microtubule assembly in neurons. We examined theexpression of all Nek kinases in adult mouse brain and detectedhigh levels of mRNA encoding Nek1, Nek3, Nek6, Nek7 and Nek9(data not shown). We also found high expression of these Nekkinases in glia-free dorsal root ganglion (DRG) sensory neuroncultures, indicating that these kinases are expressed in post-mitoticneurons (Fig. 1A).

To examine the potential impact of Nek kinases in post-mitoticneurons, we focused on Nek3 because of its high neuronalexpression and its lack of involvement in cell cycle progression orcentrosome function (Tanaka and Nigg, 1999). To furthercharacterize Nek3 expression in the nervous system, we examinedbrain lysates from embryonic and postnatal mice by westernblotting. We found that Nek3 was highly expressed duringembryogenesis and early postnatal life, but was expressed at lowerlevels in adult mice (Fig. 1B), indicating it has an important roleduring early brain development. Furthermore, Nek3 was detectedin lysates made from primary cultured DRG and hippocampalneurons (Fig. 1C).

The subcellular distribution of Nek3 in neurons was examinedusing immunohistochemistry and western blot. Similarly to previousreports using 3T3 fibroblasts, we found that endogenous Nek3 wasprimarily located in the cytoplasm of DRG neurons (Fig. 1E). Todetermine whether Nek3 is present in axons, we analyzed lysatesfrom distal axons compared with neuronal cell bodies from mouseDRG explants cultured for 14 days in vitro (DIV), a time whenaxonal extension allows for a complete separation of distal axonsfrom cell bodies. We found that Nek3 was present at similar levelsin both compartments (Fig. 1D), indicating that it is transported

into axonal processes. To further examine Nek3 distribution, weused lentivirus infection to express Myc-tagged Nek3 (Myc-Nek3)in cultured hippocampal and DRG neurons. Using anti-Mycimmunofluorescence, Nek3 was detected in cell bodies and theaxonal shaft, and strong focal Nek3 signals were detected in growthcones (Fig. 1F).

Nek3 is constitutively phosphorylated within the PEST motif atThr475Within the Nek3 C-terminal PEST domain, Thr475 is highlyconserved and is phosphorylated in embryonic mouse brain(http://phospho.elm.eu.org) (Ballif et al., 2004). To examine thephosphorylation of this residue directly, we first immunoprecipitatedMyc-tagged wild-type Nek3 and a phosphorylation-defective mutant[Nek3(T475A)] (as a control) from transfected HeLa cells andanalyzed the phosphorylation of these proteins by western blottingusing anti-phosphorylated threonine (Thr-P) antibody. We readilydetected phosphorylated wild-type Nek3 but no signal was obtainedwith Nek3(T475A) (Fig. 2A). To further examine thephosphorylation of Nek3 at Thr475, we raised antibodies against aNek3 peptide containing this phosphorylated Thr residue, and tested

Fig. 1. Nek3 is expressed in neuronal cells and is a cytoplasmic enzyme.(A) The transcripts of Nek1, Nek3, Nek6, Nek7 and Nek9 are readily detectedby RT-PCR in purified mouse DRG neurons cultured for 14 days. (B) Lysatesof brain isolated from mice of the indicated age examined by western blottingusing anti-Nek3 antibodies (BD Transduction Labs) to determine Nek3 levels.(C) Western blot showing that Nek3 is present in glia-free neuronal culturesfrom DRG or hippocampus. DIV, days in vitro. (D) Western blot of lysatesprepared from whole DRG (Total), cell bodies after axotomy (Body) andsevered axons (Axon). Schematic diagram of a single DRG neuronal ganglionwith red box depicting the site of axotomy is shown on the left.(E) Immunofluorescent microscopy of Nek3 in E15 DRG. Endogeneous Nek3is primarily located in the cytoplasm. Red, Nek3; Blue, nucleus counterstainedwith Hoechst dye 33258. (F) Immunofluorescent microscopy with anti-Mycantibody of cultured hippocampal and DRG neurons expressing Myc-taggedwild-type Nek3. Nek3 is present in the neuronal soma, axonal shaft andgrowth cones.

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against lysates of Nek3-transfected cells (Fig. 2B). This antiserarecognized only Nek3 molecules with a Thr at position 475. Toconfirm that the Nek3 Thr475-P antibody specifically recognizedNek3 phosphorylated at this residue, we treated wild-type Nek3with calf intestinal alkaline phosphatase (CIP). The anti-Thr475-Psignal detected in wild-type Nek3 was lost following CIP treatment(Fig. 2C). Next, we performed a peptide competition study, in whichthe antibody was pre-incubated with the Nek3 phosphopeptide usedas an immunogen or its non-phosphorylated counterpart.Preincubation with the Nek3 peptide containing Thr475-P resultedin a loss of reactivity with Nek3, whereas incubation with the non-phosphorylated peptide did not (Fig. 2D).

Autophosphorylation is an important regulatory step for manykinases and has been observed in several NIMA-family kinases (Fryet al., 1999; Fry et al., 1995; Graf, 2002; Helps et al., 2000; Hollandet al., 2002; Lu et al., 1993; Roig et al., 2002). To determine whetherThr475 is phosphorylated in this manner, we expressed Nek3 inHeLa cells and found that this residue was phosphorylated even ina Nek3 mutant (i.e. kdNek3) that lacks intrinsic kinase activitybecause of an Asp143 to Ala mutation in the kinase domain (Tanakaand Nigg, 1999) (Fig. 2B), indicating that this residue is unlikelyto be modified by autophosphorylation. This Thr residue conformsto a casein kinase II (CKII) consensus site (http://scansite.mit.edu/)(Obenauer et al., 2003), and we attempted to demonstratephosphorylation of Nek3 by CKII, but were unsuccessful (data notshown). Nek3 Thr475 is clearly phosphorylated by another kinase,but its identity remains unknown.

Nek3 mutation affects neuronal morphology and polarityNek kinases often contain PEST sequences, a motif associated withregulatory functions and/or protein stability. For example, the PEST

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domain of fission yeast NIMA homologue, fin1, controls its proteinlevel (and thus function) during the cell cycle (Krien et al., 1998).Similarly, removal of the PEST domain in Aspergillus NIMA alsoresulted in a stable protein that promotes a lethal prematurecondensation of chromatin (O’Connell et al., 1994). Inspection ofNek3 sequence revealed two PEST domains in the Nek3 C-terminus (PEST scores, + 9.21 N-terminal, +14.64 C-terminal;threshold of significance, +5.0) (Fig. 3A) (https://emb1.bcc.univie.ac.at/toolbox/pestfind/) (Rogers et al., 1986). To assess therole of Nek3 in neurons and the importance of Nek3 PEST domains,Myc-tagged Nek3 truncation mutants lacking either one (Nek3-T1)or both (Nek3-T2) PEST domains were generated and expressedvia lentivirus infection in DRG neurons (Fig. 3A). DRG neuronsexpressing Myc-tagged Nek3 (WT), Nek3-T1, or Nek3-T2 wereexamined for alterations in neuronal morphology byimmunohistochemistry using anti-Myc antibody 5 days afterinfection. We found that both wild-type and mutant Nek3 werelocalized to the neuronal cytoplasm and processes; however,although neurons expressing wild-type Nek3 appeared normal, thoseexpressing Nek3 truncation mutants were distinctly abnormal, withan aberrant neuronal soma and multiple neurite-like outgrowths (Fig.3B). These results indicate that Nek3 influences neuronalmorphology and that the PEST domains have important roles inregulating Nek3 protein activities.

Within the C-terminal PEST domain, Thr475 is highly conservedand was found to be phosphorylated in mouse brain (Ballif et al.,2004) and in cultured cells (Fig. 2). As protein activities are oftenregulated by post-translational modification, we explored whetherphosphorylation of Thr475 regulates Nek3 function. To this end,we generated a Nek3 mutant in which Thr475 was changed toaspartic acid [Nek3(T475D)] to mimic Thr-P. DRG neuronsexpressing Nek3(T475D), similarly to those expressing wild-typeNek3, appeared morphologically normal; however, those expressingNek3(T475A) showed cell body deformation and multiple processessimilar to those expressing Nek3 PEST domain truncation mutants

Fig. 2. Nek3 is constitutively phosphorylated at Thr475. (A) Lysates fromHeLa cells expressing Myc-tagged wild-type Nek3 or Nek3(T475A) wereimmunoprecipitated with Myc antibody and immunoblotted with antibodies tophosphothreonine (pThr) or Nek3. Only wild-type Nek3 was detected with theThr-P antibody. (B) Lysates from HeLa cells expressing indicated Nek3proteins were probed with Nek3 Thr475-P-specific antibody (pNek3Thr475).Only wild-type Nek3 and kdNek3 are recognized by this antibody. (C) HeLacell lysates containing wild-type Nek3 incubated with calf intestinal alkalinephosphatase (CIP) and analyzed by western blot. (D) Peptide competitionassay to assess Nek3 Thr475-P-specific antibody specificity. Lysate fromHeLa cells expressing wild-type Nek3 was analyzed by western blotting usingNek3 Thr475-P-specific antibody that had been preincubated with Nek3phosphorylated peptide (the immunogen), its non-phosphorylated counterpart,or no peptide (control). No signal is detected when the Nek3 Thr475-P-specific antibody was preincubated with the phosphorylated peptide.

Fig. 3. Nek3 activity is regulated by the PEST domain and phosophorylationof Thr475. (A) Schematic diagram depicting important features of Nek3 andthe constructs used in this study. Empty rectangle represents the conservedkinase domain of NIMA-related proteins; Filled boxes in C-terminus are PESTdomains; single asterisk represents Thr475 and double asterisks, Asp143, inthe ATP-binding site required for kinase activity. (B) Immunofluorescenceimages of cultured DRG neurons expressing Myc-tagged wild-type Nek3,Nek3-T1, Nek3-T2, Nek3(T475A) and Nek3(T475D) stained with anti-Mycantibody. All Nek3 proteins were located within the cytoplasm. Neuronsexpressing Nek3-T1, Nek3-T2 and Nek3(T475A) show distorted cellularmorphology and aberrant neurite outgrowth (multiple processes).

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(Fig. 3B). These results suggest that phosphorylation of Thr475within the PEST domain is a crucial step in regulating Nek3 activity,and supports the idea that the PEST domains participate in Nek3processes that regulate neuronal morphology.

A key aspect of morphology in neurons is neuronal polarity.Because Nek3 truncation mutants and a Nek3 phosphorylationdefective mutant induce abnormal neuronal morphology, weanalyzed their effects on neuronal polarity in cultured hippocampalneurons, a well characterized system for studying neuronal polarity(Dotti et al., 1988). In normally polarized hippocampal neurons,only one axonal process is present, whereas multiple axons areevidence of disturbed neuronal polarity. We infected these neuronswith lentiviruses expressing Nek3, Nek3(T475A), Nek3-T1 orNek3-T2 and stained them 3 days later with neuron-specific β IIItubulin (Tuj1) antibody to analyze their processes and morphology.Control neurons and those expressing wild-type Nek3 bore only asingle long axon, characteristic of a normally polarized hippocampalneuron (Fig. 4A). By contrast, Nek3(T475A) and Nek3 truncationmutants induced the growth of multiple long processes. The identityof these processes as axons was verified by staining with the axonalmarker Tau-1 (Fig. 4B), thus demonstrating that Nek3 mutationdisturbed the polarity of these neurons.

To determine whether the effects of the Nek3 mutants onneuronal morphology and polarity required kinase activity, wegenerated additional mutants in which a crucial residue in the kinasedomain, Asp143, was mutated to Ala to produce a catalytically deadNek3 protein (Fig. 3A) (Tanaka and Nigg, 1999). This mutation(D143A) was engineered into the Nek3-T2 mutant to create kinase-dead Nek3-T2 (kdNek3-T2). This mutant was expressed along withNek3-T2 in hippocampal neurons and analyzed as above. We foundthat neuronal abnormalities induced by Nek3-T2 were not present

in neurons expressing kinase-dead Nek3-T2 (Fig. 4A,C), indicatingthat kinase activity is required for mutant Nek3 to induceabnormalities in neuronal morphology and polarity. The observationthat these Nek3 mutant-mediated alterations in morphology andpolarity require intact kinase activity further indicates that theseeffects are not caused by non-specific effects due to mis-foldedmutant Nek3.

Nek3 mutants alter levels of acetylated α-tubulinPost-translational modifications of microtubules, such as acetylation,tyrosination, phosphorylation and others, have crucial roles inestablishing and maintaining neuronal polarity and morphology(Fukushima et al., 2009). For example, reducing the level ofmicrotubule acetylation causes abnormalities in dendritic branchingof cultured neurons (Ohkawa et al., 2008) and branch formation ofcortical neurons in the developing mouse brain (Creppe et al., 2009).The effects of Nek kinases on microtubule dynamics during mitosisand ciliogenesis (O’Regan et al., 2007; Quarmby and Mahjoub, 2005)along with our observation that Nek3 mutants affect neuronalmorphology and polarity prompted us to examine whether they mightalso alter neuronal microtubule acetylation. DRG neurons expressingwild-type Nek3, Nek3(T475A), kdNek3(T475A), Nek3-T2 andkdNek3-T2 were analyzed with an antibody specific for acetylatedα-tubulin (Ac-tubulin), a marker of stable microtubules (Cambray-Deakin and Burgoyne, 1987; Schulze et al., 1987). We found thatneurons expressing Nek3-T2 and Nek3(T475A) had greatly reducedlevels of Ac-tubulin (Fig. 5A). However, Nek3 mutants lacking kinaseactivity had normal levels of Ac-tubulin, again indicating the necessityfor kinase activity in these Nek3-mutant-mediated effects.

The cellular microtubular network is particularly well visualizedin HeLa cells; therefore, to further analyze the effects of Nek3 on

Fig. 4. Nek3 mutants disrupt neuronal polarity. (A) Hippocampal neurons infected with lentivirus expressing EGFP, wild-type Nek3 or Nek3 mutants were stainedwith neuron-specific β III tubulin (Tuj1) antibody to highlight their morphology. A single long process was observed in neurons expressing EGFP, wild-type Nek3or kinase-dead (kd)Nek3-T2. However, neurons expressing Nek3(T475A), Nek3-T1 or Nek3-T2 have multiple long processes indicative of aberrant polarity.(B) Immunohistochemical analysis using the Tau-1 axonal marker demonstrating that the multiple processes induced by Nek3-T2 are axons (arrows; cell body isdenoted by arrowhead). In addition, the Tau-1-stained processes display morphology typical of axons with a very fine caliber near the nucleus and graduallyincreasing caliber toward the end. (C) Quantification of hippocampal neuronal polarity. Neurons were cultured as above and the number of processes per neuronwere counted. Approximately 200 neurons expressing each of the indicated proteins were analyzed per experiment (n=3 independent experiments). Neuronsextending a single axon were defined as polarized. Nek3(T475A), Nek3-T1 and Nek3-T2 induced aberrant polarity in hippocampal neurons; however, an activekinase was required to induce the formation of multiple processes (see kdNek3-T2). Results are mean ± s.d. *P<0.0001 (vs EGFP control).

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the Ac-tubulin network, we analyzed HeLa cells expressing wild-type Nek3, Nek3(T475A) or kdNek3(T475A). Immunofluorescentmicroscopy showed that HeLa cells expressing Nek3(T475A),similarly to DRG neurons, had a markedly diminished Ac-tubulincytoplasmic network (Fig. 5B). Decreased levels of Ac-tubulin werealso observed in cells expressing Nek3-T1 or Nek3-T2, but nochanges were seen in cells expressing wild-type Nek3 or kinase-dead versions of the Nek3 mutants (Fig. 5B, data not shown). Wealso noted the absence of cilia in cells expressing Nek3(T475A) orNek3-T2, but not kdNek3-T2 or wild-type Nek3 (supplementarymaterial Fig. S1), suggesting that the Nek3 mutation is detrimentalto cilia integrity. In contrast to levels of Ac-tubulin, the overallpattern of the microtubule network highlighted by anti-α-tubulinimmunofluorescence was not affected by Nek3(T475A) (Fig. 5C),indicating that Nek3(T475A) affects the acetylation of α-tubulin,but not the α-tubulin network per se.

In addition to diminished levels of Ac-tubulin, HeLa cellsexpressing Nek3(T475A) or Nek3 truncation mutants assumed ahighly elongated shape and became aligned with neighboring cells(Fig. 6A). Interestingly, the cells expressing Nek3(T475A) appearedeven more deformed at 72 hours, with neurite-like projections (datanot shown). Notably, phalloidin staining, which detects polymerizedactin, demonstrated that these Nek3-mutant-mediated morphologicalalterations were not accompanied by changes in the actin filamentnetwork (data not shown). Furthermore, no changes in proliferationwere induced by expression of wild-type or mutant Nek3 (data notshown), which is consistent with previous reports that Nek3 doesnot directly affect cell cycle progression (Tanaka and Nigg, 1999).

The alterations in α-tubulin acetylation could drive the changesin neuronal morphology and polarity induced by Nek3 mutation.Two major effectors of α-tubulin acetylation state are thedeacetylases, HDAC6 (Hubbert et al., 2002; Matsuyama et al., 2002)and SirT2 (North et al., 2003). To explore whether they areinvolved in Nek3-mutant-mediated decreases in Ac-tubulin, wetreated HeLa cells expressing Nek3(T475A) with nicotinamide, a

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sirtuin inhibitor, trichostatin A (TSA), a broad-spectrum HDACinhibitor, or tubacin, an HDAC6-specific inhibitor (Haggarty et al.,2003). After 24 hours, immunofluorescence microscopy was usedto monitor the levels of Ac-tubulin. We found that Ac-tubulin levelsremained very low in cells expressing Nek3(T475A) treated withnicotinamide (a SirT2 inhibitor) (North et al., 2003), indicating thatsirtuins are not involved in this deacetylation process [Fig. 6B;compare Ac-tubulin levels in cells denoted by arrow (transfected)and asterisk (non-transfected) in nicotinamide-treated cells]. Thisis very similar to DMSO-treated cells, where Nek3(T475A)-expressing cells showed greatly diminished Ac-tubulin. However,addition of either TSA or tubacin, restored microtubule acetylationin Nek3(T475A)-transfected cells to levels present in non-transfected cells [Fig. 6B; compare Ac-tubulin levels in cells denotedby one asterisk (transfected) and two asterisks (non-transfected) intubacin- or TSA-treated cells]. These results clearly indicate thatHDAC6 activity is required for loss of Ac-tubulin observed in cellsexpressing mutant Nek3.

DiscussionNek kinases were initially studied for their roles in regulating cilia,centrosomes and microtubule dynamics during cell cycle. Thediscovery that some Nek kinases are expressed in the adult brainraised the possibility that they participate in additional, non-mitoticroles in neurons. In Chlamydomonas, a NIMA-family kinase iscrucial for cilia severing because it promotes the disassembly ofmicrotubules as cells progress towards mitosis (Bradley et al., 2004;Finst et al., 2000; Finst et al., 1998; Fliegauf et al., 2007; Mahjoubet al., 2002; Mahjoub et al., 2004). Given the similarities betweencilia and neuronal axons (both are microtubule-rich structuresextending from the cell body) and the role of Nek kinases inmicrotubule regulation, we initiated studies to test whethermammalian Nek kinases regulate microtubules in neurons. We foundthat Nek3, which is not involved in mitosis, was expressed at highlevels in developing mouse brain and in cultured post-mitotic

Fig. 5. Nek3 mutants induce α-tubulin deacetylation.(A) The level of α-tubulin acetylation in lysates fromDRG neurons expressing the indicated Nek3 proteinswas analyzed by western blotting using anti-Ac-tubulinantibody. Neurons expressing Nek3-T2 andNek3(T475A) have low levels of Ac-tubulin. Total α-tubulin (α-tub) levels are equivalent in all samples. Mycantibody was used to examine Nek3 protein levels.(B) HeLa cells transfected with constructs expressingwild-type Nek3, Nek3(T475A) or kdNek3(T475A) andstained with anti-Ac-tubulin (red). EGFP signal indicatestransfected cells (green). An untransfected cell withnormal acetylated microtubule network is indicated withan arrow. Nek3(T475A)-transfected cells show analmost complete lack of acetylated microtubules(arrowhead and asterisk). Cells expressing wild-typeNek3 or kdNek3(T475A) are indistinguishable fromuntransfected cells. Insert is an enlarged view of theacetylated microtubule network in untransfected andNek3(T475A)-transfected cells. (C) HeLa cellstransfected with Nek3(T475A) stained for total α-tubulin (red) to examine the microtubule networkindependent of its acetylation state. EGFP (green)indicates transfected cells. The area of the enlarged view(insert) is indicated by an asterisk. Note that mostuntransfected cells have a rounded morphology, whereasT475A-transfected cells display an elongated shape. Nodifferences in the microtubule network were observed intransfected versus untransfected cells (insert).

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neurons. The expression of Nek3 mutants lacking a key regulatoryphosphorylation site (Thr475) caused changes in cellularmorphology and polarity of both primary cultured central andperipheral neurons as well as epithelial cells. These cellular changesinduced by Nek3 mutations are dependent on intact kinase activity,because Nek3 mutant versions with an additional mutation in theactive kinase site itself did not produce these cellular changes.Interestingly, Nek3 mutants with an intact kinase domain alsostimulated dramatic increases in α-tubulin deacetylation that wereconsistent with the effects of reduced microtubule stability, such asthat induced by NIMA-family kinase during deflagellation inChlamydomonas.

Cells overexpressing PEST truncated or phosphorylation-defective (Thr475) Nek3 mutants have dramatic alterations in theirmicrotubule cytoskeleton and greatly reduced acetylatedmicrotubules in the cytoplasm. These changes are strikingly similar

to those induced by knocking down another cilia-related proteinBBIP10 (Loktev et al., 2008). BBIP10 is a subunit of a stable proteincomplex, the BBSome, that participates in the function and vesiculartrafficking of the primary cilium (Berbari et al., 2008; Nachury etal., 2007). Similarly to the NIMA-family kinases, components ofthe BBSome have been proposed to be important for microtubuleand centrosome-based functions, because mutations in BBSomeproteins underlie a variety of genetic disorders referred to asciliopathies (Badano et al., 2006). The strikingly similar phenotypesinduced by overexpressed mutant Nek3 and loss of BBIP10 raisethe possibility that Nek3 and BBSIP10 could negatively regulateeach other and influence the same pathways. Indeed, in addition toa marked reduction in cytoplasmic microtubule acetylation, NIH3T3cells expressing the Nek3(T475A) mutant also show dramaticdecreases in Ac-tubulin signal for cilia, which again is similar tocells lacking BBIP10 (Loktev et al., 2008). Other connectionsbetween Nek kinases and BBS proteins include centrosome splitting,which occurs in cells overexpressing Nek2 (Fry et al., 1998) orlacking BBS8 (Loktev et al., 2008). Better understanding of theinterplay between Nek kinases and BBS proteins might lead tofurther insights into ciliary function and its role in disease.

Structure-function experiments revealed that the C-terminalPEST domains and, in particular, the phosphorylation of Thr475within the PEST domain were critical modulators of Nek3 function.Nek3 mutants lacking these motifs or defective in Thr475phosphorylation stimulated a signaling pathway that ultimately ledto the activation of HDAC6 and α-tubulin deacetylation, a processthat leads to reduced microtubule stability (Cambray-Deakin andBurgoyne, 1987; Matsuyama et al., 2002; Schulze et al., 1987; Tranet al., 2007). Interestingly, HDAC6 has also been implicated in ciliaresorption or disassembly (Pugacheva et al., 2007) and α-tubulindeacetylation induced by depletion of BBIP10 (Loktev et al., 2008).How the PEST domain or the dephosphorylation of Thr475 regulatesNek3 function is an important question. The PEST domain wasinitially identified as a protein motif that promoted rapid proteindegradation (Rogers et al., 1986); however, it is now known to alsomediate protein-protein interactions (Pongubala et al., 1992; Zhanget al., 2007). Removal of the PEST domain from NIMA-familykinase in fission yeast resulted in elevated activity because ofincreased protein stability and higher protein levels (Krien et al.,1998; O’Connell et al., 1994). However, the truncation of the PESTdomains in Nek3 led to decreased levels of protein, indicating thatincreased Nek3 levels are not responsible for the abnormalitiescaused by these mutants. The Nek3 PEST truncation mutantsproduced abnormalities that were identical to those induced byNek3(T475A), suggesting that they both adopt a similar proteinconformation. Based on the abnormalities induced by these mutantsand on the predicted structural flexibility of the Nek3 PEST regions(http://imtech.res.in/raghava/apssp/), we propose that these domainsmodulate Nek3 activity through an allosteric effect that alters inter-or intramolecular interactions depending on the phosphorylationstate of Thr475 within the PEST domain.

Acetylation of microtubules is critical for neuronal developmentand function. For example, increased microtubule acetylation viainhibition of Sirt2 results in decreased α-synuclein-mediatedneurotoxicity (Outerio et al., 2007) and HDAC6 inhibitors reversetransport deficits in neurons expressing mutant Huntington diseaseprotein (Dompierre et al., 2007). By contrast, decreased microtubuleacetylation has been found to lead to abnormal neuronal shape andmigration (Creppe et al., 2009; Ohkawa et al., 2008). Similarly, wefound that neurons expressing mutant Nek3 also have dramatic

Fig. 6. Nek3-regulated α-tubulin deacetylation requires HDAC6. (A) HeLacells expressing EGFP, wild-type Nek3 or Nek3(T475A) stained for Ac-tubulin 48 hours after transfection. Note that cells expressing Nek3(T475A)have reduced Ac-tubulin levels and are configured in an elongated, alignedmanner. Cells expressing wild-type Nek3 are indistinguishable from controlcells expressing GFP. (B) Nek3(T475A)-expressing HeLa cells treated withtrichostatin A (TSA) (500 nM), tubacin (20 μM), nicotinamide (20 mM) orDMSO (control) and stained with anti-Ac-tubulin antibody to assess the levelof acetylated microtubules. DMSO- and nicotinamide-treated cells display thecharacteristic robust reduction in microtubule acetylation mediated byNek3(T475A) (compare cells denoted by arrow and asterisk in DMSO- andnicotinamide-treated cells). Cells treated with the broad HDAC inhibitor TSAor the HDAC6-specific inhibitor tubacin have normal levels of acetylated α-tubulin [compare adjacent untransfected cell (two asterisks) withNek3(T475A)-expressing cell (one asterisk)], indicating that these inhibitorsrescue the phenotypes induced by Nek3(T475A). All images were taken withthe same exposure time.

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abnormalities in neuronal polarity and morphology as well asreduced levels of microtubule acetylation. Although the mechanismresponsible for these abnormalities is still unknown, increasedmicrotubule acetylation is commonly associated with their stability,thus decreased microtubule stability could be responsible for theneuronal abnormalities caused by Nek3 mutants.

Nek3 can also regulate prolactin-stimulated actin-mediated cellmobility through Vav1 or Vav2 and Rac1 activation in breast cancercells (Miller et al., 2007; Miller et al., 2005). However, we foundthat Nek3 mutants could still induce morphological alterations inneurons derived from Vav1-Vav2-Vav3 triple-deficient mice(unpublished data), seemingly in contradiction to these experiments.Furthermore, in contrast to results in breast cancer cells, Nek3-induced alterations in neurons occur through microtubule alterations,with no evidence for changes in actin polymerization. Finally,kdNek3 expression causes apoptosis in breast cancer cells (Milleret al., 2005), but Nek3-regulated increases in neuronal cell deathwere not observed in our experiments.

In summary, we discovered that the PEST domains and Thr475are critical for Nek3 activity and that Nek3 affects neuronalmorphology and polarity. These effects are presumably mediatedby Nek3 mutant stimulation of HDAC6 α-tubulin deacetylation.The presence of Nek3 in axons and its ability to regulate microtubuleacetylation suggests that it could be involved in processes linkedto axonal projection and degeneration. The identification of thekinase(s) that regulate Nek3 through phosphorylation of Thr475,as well as characterization of the downstream substrates of Nek3,will help shed new light on its role in the nervous system.

Materials and MethodsAntibodiesAntibodies were routinely used at a 1:1000 dilution unless otherwise stated. Anti-Myc (9B11) (Cell Signaling 2276), anti-Nek3 (BD Transduction Laboratories611716; dilution 1:250), anti-β-tubulin (Developmental Studies hybridoma; dilution1:5000), anti-phosphothreonine (42H4) (Cell Signaling 9386), Nek3 Thr475-P (inhouse; dilution 1:1000 from 200 μg/ml), anti-Tau-1 (Chemicon MAB3420), anti-neuronal class III B-tubulin (TujI) (Covance PRB-435P), anti-α-tubulin (B5-1-2)(Sigma T5168), anti-acetylated α-tubulin (6-11B-1) (Sigma T6793). All secondaryantibodies were from Jackson ImmunoResearch. Rabbit IgG-Cy3 (111-166-045),mouse IgG-Cy3 (115-165-003), rabbit IgG-HRP (111-035-144), mouse IgG-HRP(115-035-003).

Plasmid and lentivirus productionFull-length mouse Nek3 cDNA was obtained from Open Biosystems (Huntsville,AL) and a Myc tag was added to the N-terminus after the initiator ATG using PCR.Nek3 mutants were generated by PCR mutagenesis, cloned into FUIV (Araki et al.,2004), a lentiviral vector containing the human ubiquitin promoter along with EGFPcassette expressed via an IRES so that infected cells can be identified. Lentiviruswas produced as described previously (Araki et al., 2004).

Primary neuronal cultures and lentivirus infectionDissociated dorsal root ganglia (DRG) neurons from E13 mouse embryos werecultured in Neurobasal medium with B27 (Invitrogen) as described previously (Chenet al., 2008). The medium contained the anti-mitotic reagent 5-Fluoro-2�-deoxyuridine(FDU) (Sigma) and uridine (Sigma) at final concentration of 20 μM for the first 3days to eliminate proliferating, non-neuronal cells. Hippocampal neurons were isolatedfrom E15 embryos and cultured as above, except no FDU was added to these cultures.Neurons were infected with lentiviruses at the time of plating for hippocampal culturesand 3 days after plating for DRG cultures using ~107 colony-forming units (CFU)/mlas previously described (Araki et al., 2004). More than 95% of the neurons wereinfected using this procedure.

Immunoblotting and immunofluorescenceCells were lysed on ice for 30 minutes in NEB buffer (50 mM HEPES-KOH, pH7.5, 5 mM MnCl2, 10 mM MgCl2, 2 mM EDTA, 100 mM NaCl, 5 mM KCl, 0.1%NP-40, 20 mM β-glycerophosphate, 20 mM sodium fluoride, 2 mM sodiumorthovanadate and proteinase inhibitor from Roche) and lysates were clarified bycentrifugation at 13,000 � g for 10 minutes at 4°C. Western blotting was performedfollowing standard procedures. For immunofluorescence, neurons or HeLa cells grown

on coverslips (Carolina; ww-63-3029) were fixed with 4% paraformaldehyde in PBSpH 7.4 for 20 minutes. The cells were permeabilized/blocked by incubation in 0.3%Triton X-100, 10% goat serum (Invitrogen) in PBS for 1 hour at room temperature.Cells were then incubated with the indicated primary antibody in 0.1% Triton X-100, 10% goat serum in PBS for 16 hours at 4°C. The signals were visualized byincubation with Cy3-conjugated fluorescent secondary antibodies in 0.1% Triton X-100, 10% goat serum in PBS for 1 hour at room temperature.

Nek3 Thr475-P antibody productionTo produce antibodies specific for Nek3 phosphorylated at Thr475, rabbits wereimmunized with peptides corresponding to Nek3 residues 466-481 [Cys-FEPRLDEEDT(-P)DFEEDN, where T(-P) represents phosphothreonine] at PacificImmunology. The anti-Nek3 antisera were sequentially affinity purified usingSulfoLink Coupling Gel (Pierce 20401) conjugated with non-phospho-peptide (Cys-FEPRLDEEDTDFEEDN) and phospho-peptide according to the manufacturer’sprotocol.

Cell culture and transfectionHeLa cervical carcinoma cells were grown in Dulbecco’s modified Eagle’s medium(Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 100 IU/mlpenicillin, 100 μg/ml streptomycin and 20 mM L-glutamine at 37°C in a 5% CO2

atmosphere. Transfections were performed using FuGENE 6 (Roche Diagnostics)according to the manufacturer’s instructions (1:3 of plasmid and FuGene reagentratio was used). About 80-90% cells were transfected. Where indicated, the cellswere treated 48 hours after transfection with nicotinamide (20 mM), tubacin (20 μM)or TSA (500 nM) for the indicated time.

In vitro phosphatase assay and peptide competition assayLysates of HeLa cells expressing Myc-Nek3 were prepared, clarified by centrifugationand incubated with 20 U calf intestinal phosphatase (CIP) for 1 hour in therecommended buffer (New England Biolabs). Reactions were stopped by additionof 6� Laemmli buffer and the lysates were analyzed by western blotting. For peptidecompetition assays, the Nek3 Thr475-P antibody was incubated with a fivefold molarexcess of the respective peptide before use in western blotting.

Statistical analysisStatistical analysis was carried out using two-way ANOVA using Graphpad Prism.

We thank Ta-Chiang Liu, Yo Sasaki, Jason Gustin and Bin Zhangfor reading this manuscript. This work was supported by NIHNeuroscience Blueprint Center Core Grant P30 NS057105 toWashington University, the HOPE Center for Neurological Disorders,and National Institutes of Health Grants CA111966, NS040745,AG13730 (to J.M.), and MDA grant 3972 (J.M.). R.H.B. is supportedby grants from the NIH (K08NS055980) and the Muscular DystrophyAssociation and holds a Career Award for Medical Scientists from theBurroughs Wellcome Fund. Deposited in PMC for release after 12months.

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