Unique Post-Translational Modifications in Specialized Microtubule Architecture

Koji Ikegami∗ and Mitsutoshi Setou Department of Molecular Anatomy, Molecular Imaging Advanced Research Center, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan

ABSTACT. Microtubules (MTs) play specialized roles in a wide variety of cellular events, e.g. molecular transport, cell motility, and cell division. Specialized MT architectures, such as bundles, axonemes, and centrioles, underlie the function. The specialized function and highly organized structure depend on interactions with MT-binding proteins. MT-associated proteins (e.g. MAP1, MAP2, and tau), molecular motors (kinesin and dynein), plus-end tracking proteins (e.g. CLIP-170), and MT-severing proteins (e.g. katanin) interact with MTs. How can the MT-binding proteins know temporospatial information to associate with MTs and to properly play their roles? Post-translational modifications (PTMs) including detyrosination, polyglutamylation, and polyglycylation can provide molecular landmarks for the proteins. Recent efforts to identify modification- regulating enzymes (TTL, carboxypeptidase, polyglutamylase, polyglycylase) and to generate genetically manipulated animals enable us to understand the roles of the modifications. In this review, we present recent advances in understanding regulation of MT function, structure, and stability by PTMs.

Key words: microtubule/post-translational modification/detyrosination/glutamylation/glycylation

Introduction between MT and a variety of its binding partners. The molecules contain various proteins with a wide range of mass, The microtubule (MT) is one of three major cytoskeletons structure, and function. Molecular motor proteins, kinesins highly conserved in eukaryotes. As the name indicates, the and dyneins, use MT as a molecular railway and walk on it. MT has a tubular structure with a diameter of 25 nm (Fig. MT-associated proteins (MAPs) starch up the MTs, and 1). The tubule wall is composed of thirteen protofilaments, thus stabilize them. Plus-end tracking proteins (+TIPs) which is further constructed by heterodimers of two small accumulate on the distal tip of the MT. Some other molec- globular proteins, α- and β-tubulins (Mohri, 1968) (Fig. 1). ular species can sever and thus destabilize MT. Those MT- MTs play specific roles in a variety of cellular events, which interacting proteins associate with MTs in designated sub- range from directional molecular transport, ciliary or flagellar cellular positions to play their roles properly. motility, and chromosome segregation, to cytokinesis. To What leads the MT-interacting proteins to their work play these specialized roles, the MT constructs higher-order place and how are they regulated, as different kinesin architecture in related subcellular compartments, such as motors deliver their cargos to each specific destination in a bundles (in neuronal processes), axonemes (in flagella and neuronal cell (Setou et al., 2000; Setou et al., 2002; Setou et cilia), and centrioles (in basal bodies or spindle bodies) al., 2004)? Two mechanisms can be assumed. One is a well- (Fig. 1). known regulatory system, the post-translational modifica- How does the simple tubular MT form the highly orga- tions (PTMs) of MT-interacting proteins. The most popular nized architecture? The mechanism relies on interactions example is the phosphorylation of MAPs. Evidence has accumulated that the phosphorylation of MAPs affects *To whom correspondence should be addressed: Koji Ikegami, Depart- their function. The phosphorylation of tau inhibits the func- ment of Molecular Anatomy, Molecular Imaging Advanced Research tion of tau to promote MT assembly (Nishida et al., 1982; Center, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan. Yamamoto et al., 1983; Lindwall and Cole, 1984; Hoshi et Tel: +81–53–435–2085, Fax: +81–53–435–2292 al., 1987; Wada et al., 1998). The phosphorylation of MAP2 E-mail: [email protected] counteracts MAP2-mediated microtubule polymerization Abbreviations: mAb, monoclonal antibody; MAP, microtubule-associated protein; MT, microtubule; PTM, post-translational modification; TTL, (Jameson et al., 1980; Nishida et al., 1981; Yamamoto et tubulin tyrosine ligase. al., 1983; Nishida et al., 1987; Hoshi et al., 1988). Hence,

15 K. Ikegami and M. Setou

Fig. 1. Microtubule in cell. Microtubule-enriched subcellular compartments or structures are depicted. Microtubule is shown in red. Microtubule- constructed higher architectures, such as bundle, axoneme, and centriole, are highlighted in boxes. Individual microtubule with diameter of 25 nm is composed of 13 protofilaments, which is further constructed from multiple heterodimers of α- and β-tubulins. PTMs currently known are listed below. phosphorylation of MAPs negatively regulates MTs dynam- review the currently available evidence for the roles of the ics and stability (Drewes et al., 1997). CRMP2 phosphory- PTMs in mammals with emphasis on recent progress. We lation inactivates the activity of CRMP2 to promote micro- also present some evidence obtained from genetically modi- tubule assembly and controls neuronal polarity (Yoshimura fied non-mammalian organisms. Readers are referred to et al., 2005). Moreover, MAPs phosphorylation indirectly some review articles for other general modifications (acety- modulates kinesin motor trafficking through affecting lation, phosphorylation, and palmitoylation) (Rosenbaum, MAPs-MT binding affinity (Sato-Harada et al., 1996). 2000; Westermann and Weber, 2003; Verhey and Gaertig, The multi-microtubule hypothesis can support another 2007; Hammond et al., 2008). mechanism. The heterogeneities of tubulin molecules underlie the model (Kobayashi and Mohri, 1977). The heterogeneities are made by two independent mechanisms: Detyrosination/Tyrosination genome-encoded multi-tubulin genes and PTMs. Mamma- lian genome encodes 6 to 8 different tubulin molecules for The cycle of detyrosination and tyrosination is a PTM of each α- and β-tubulins. Those different tubulins show dif- tubulin that has been investigated for the longest time. This ferent tissue-specific expression patterns. PTMs can more PTM occurs in most α-tubulins. In this cycle, a tyrosine that drastically contribute to the heterogeneities. is encoded as the C-terminal amino acid by genome is Tubulin is subjected to the detyrosination/tyrosination removed (Argaraña et al., 1978), and a free tyrosine is re- cycle, the removal of penultimate glutamate, polyglutamy- added to the C-terminal of detyrosinated α-tubulin (Barra et lation, polyglycylation, acetylation, phosphorylation, and al., 1974). Long-living, i.e. stable MTs are enriched by palmitoylation (Fig. 1). These modifications, except for detyrosinated tubulins while highly dynamic MTs contain acetylation, occur in the C-terminal region, which surfaces abundantly tyrosinated tubulins (Wehland and Weber, on MT lattice where many MT-binding proteins interact. In 1987). A recent work demonstrates that tubulin detyrosina- this review, we focus on unique PTMs (detyrosination/ tion inhibits MT disassembly through suppressing interac- tyrosination, polyglutamylation, and polyglycylation), and tions of MT-depolymerizing motor, MCAK or KIF2 with

16 Unique Post-Translational Modifications in Specialized Microtubule Architecture

Fig. 2. Enzyme for tubulin PTMs. Identified enzymes for modifications are shown in red boldface. Carboxypeptidase for generating detyrosinated tubulin is still only a candidate (yellow). Unidentified enzymes are shown in light gray.

MTs (Peris et al., 2009), although detyrosination has been al., 2006). Indeed, CLIP-170 preferentially binds in vitro to thought not to modify MT stability for a long time (Webster tyrosinated α-tubulin (Peris et al., 2006) and to the –EEY/F et al., 1990). Detyrosinated tubulin is further subjected to motif seen in the α-tubulin C-terminal (Honnappa et al., next deglutamylation, i.e. removal of penultimate glutamate, 2006; Mishima et al., 2007). Similar results are observed in producing Δ2-tubulin (Paturle-Lafanechère et al., 1991) a most primitive eukaryote, the budding yeast. Genetical (Fig. 1). In the current model, Δ2-tubulin is thought to be deletion of phenylalanine from the C-terminal of α-tubulin excluded from the detyrosination/tyrosination cycle results in mislocalization of Bik1p, a yeast homolog of (Westermann and Weber, 2003; Rüdiger et al., 1994). CLIP-170 (Badin-Larçon et al., 2004). An enzyme that accounts for the detyrosination/tyrosina- Another line of evidence indicates that the modification tion cycle is the oldest enzyme identified as a tubulin- affects the interaction between molecular motors and MTs. modifying enzyme. The enzyme for re-addition of tyrosine Kinesin-1 shows more strong binding affinity to MT enriched to detyrosinated α-tubulin, termed tubulin tyrosine ligase by detyrosinated tubulin in vitro (Liao and Gundersen, (TTL) (Raybin and Flavin, 1977) (Fig. 2), is purified from 1998). In vivo, kinesin-1 preferentially binds to detyrosi- porcine brain (Murofushi, 1980), and the polypeptide is nated tubulin-rich MTs (Dunn et al., 2008). Kinesin-1 selec- completely identified (Ersfeld et al., 1993). In contrast, the tively accumulates in a future axon in early stage of neu- enzyme removing the terminal tyrosine is not fully identi- ronal growth (Jacobson et al., 2006). The axonal shaft is fied, while recent researches propose a cytosolic carboxy more enriched by detyrosinated tubulins than dendrites and peptidase, CCP1/Nna1 as a strong candidate (Kalinina et cell body during early neuronal differentiation (Arregui et al., 2007; Rodriguez de la Vega et al., 2007) (Fig. 2). al., 1991). In light of the knowledge that has accumulated, Enzymes performing the removal of penultimate glutamate our group has recently provided clear evidence that tubulin from detyrosinated tubulin remain to be identified. Enzymes detyrosination is a spatial cue for neuronal polarity determi- re-adding glutamate to Δ2-tubulin might be present, though nation through preferential interaction between KIF5-loop8 currently nobody suggests this idea. and detyrosinated tubulin-rich MTs (Konishi and Setou, Recent progress for understanding the physiological roles 2009). The reason why neurons cultured from the TTL-null of this modification cycle is achieved by a generation of a animal form multiple axons (Erck et al., 2005) can be TTL-null mouse line. The TTL-null animal provides evi- explained by this model, whereby kinesin-1 might fail to dence for a vital role of tyrosinated tubulin. The mouse find the future axon since MTs are uniformly detyrosinated. shows malformation of layer in cerebral cortex, disruption Such a model allows us to speculate that the neuronal of cortico-thalamic loop, and perinatal lethality along with degeneration observed in Purkinje cell degeneration (pcd) prominent loss of tyrosinated tubulins (Erck et al., 2005). mouse (Mullen et al., 1976; Landis and Mullen, 1978), a Neurons cultured from the animal display abnormal neurite spontaneous mutant of CCP1/Nna1 (Fernandez-Gonzalez et growth along with forming multiple axons (Erck et al., al., 2002), might result from inefficient intracellular trans- 2005). In the neurons, the cytoplasmic linker protein ports caused by lowered binding affinity between kinesin-1 (CLIP)-170, a +TIP containing glycine-rich cytoskeleton- and MTs that lack detyrosinated tubulins. associated protein (CAP-Gly) domains, is dispersed from the growth cone (Erck et al., 2005). Fibroblasts cultured from the TTL-null animal display mislocalization of CLIP- 170 and abnormal orientations of spindle bodies (Peris et

17 K. Ikegami and M. Setou

Fig. 3. Polyglutamylation and polyglycylation. (A) Schematic view of polyglutamylation and polyglycylation. Poly-glutamate (EEE···) or -glycine (GGG···) chain is attached to a glutamate residue (E) in a protein backbone. Note that the polyglutamate chain has negative charges. (B) More detailed diagram of polyglutamylation. The first glutamate is bound to a γ-carboxyl group of a glutamate residue. Subsequent addition of glutamates is achieved by bonds between α-carboxyl group and amino group. Note that the polyglutamate chain provides extra carboxyl groups that result in increases in total negative charges.

Polyglutamylation deficient mouse shows impaired synaptic transmission and a decrease in the amount of synaptic vesicles in synaptic Tubulin polyglutamylation was first reported in pig brain terminals associated with a gross loss of polyglutamylation MT (Eddé et al., 1990). This modification is highly intrigu- on α-tubulin in brain homogenate (Ikegami et al., 2007). ing from two points. First, the form of modification is Trafficking of KIF1A (kinesin-3 family), but neither KIF3A unique. A long chain of polypeptides constructed from mul- (kinesin-2 family) nor KIF5A (kinesin-1 family), is impaired tiple glutamates are covalently linked on a γ-carboxy group in the neurons cultured from the mouse (Ikegami et al., of glutamate residue in tubulin C-terminal region (Fig. 3). 2007). In vitro, binding of KIF1A to MT is most affected by Second, the long polyglutamate chain increases remarkably the loss of polyglutamylated α-tubulin (Ikegami et al., negative charges in the tubulin C-terminal (Fig. 3), which 2007). This KIF1-selective impairment can be explained by readily suggests the important roles of the charges in the the finding that KIF1 has a longer K-loop than KIF3 or interaction between MT and its binding partners. Studies KIF5 (Okada and Hirokawa, 1999). using a specific monoclonal antibody (mAb) GT335 against Evidence has accumulated for the spatial and temporal glutamylated tubulin reveal that polyglutamylated tubulins distribution of polyglutamylated tubulins. In neuronal cells, are enriched in neuronal cells (Wolff et al., 1992), and that polyglutamylated β-tubulin concentrates in the somato- axonemal and centriolar MTs are also highly modified dendritic region, whereas polyglutamylated α-tubulin accu- (Fouquet et al., 1994; Bobinnec et al., 1998b). mulates in the neuronal processes (Ikegami et al., 2006; A recent significant breakthrough in the area of poly- Ikegami et al., 2007) (Fig. 4). In mouse brain, polyglutamy- glutamylation research is the success in identifying lated β-tubulin emerges after birth, whereas modified α- modification-performing enzymes, polyglutamylases (or tubulin is already prominent at birth (Audebert et al., 1994). glutamate ligases) (Fig. 2). Interestingly, the enzymes are In culture, polyglutamylated β-tubulin emerges and increases members of a protein family homologous to TTL. The first in parallel with neuronal growth, while modified α-tubulin identified TTLL1 is a subunit of the polyglutamylase com- is detected immediately after starting the culture (Audebert plex purified from mouse brain (Janke et al., 2005). Further et al., 1994). The late increase in level of polyglutamylated studies demonstrate that TTLL4, 5, 6, 7, 9, 11, and 13 have β-tubulin may be involved in postnatal growth and matura- tubulin polyglutamylase activity (Ikegami et al., 2006; van tion of neuronal processes, especially dendrites. Indeed, Dijk et al., 2007; Wloga et al., 2008; Mukai et al., 2009). knockdown of TTLL7 in PC12 cells demonstrates that Currently, the glutamate-removing enzyme, deglutamylase TTLL7-mediated β-tubulin polyglutamylation is required for or glutamatase remains to be identified. CCP 2 to 6, which neurite growth (Ikegami et al., 2006). What involves poly- are members of cytosolic carboxypeptidases with similarity glutamylated β-tubulin in neurite growth currently remains to CCP1/Nna1 (Kalinina et al., 2007; Rodriguez de la Vega to be elucidated. However, MAP1A is a plausible candidate. et al., 2007), might harbor the enzymes. This hypothesis can be supported by three independent lines The identification of enzymes enables us to better under- of evidence: i) strong dependency of MAP1A-MT interac- stand the physiological roles of tubulin polyglutamylation tion on level of tubulin polyglutamylation (Bonnet et al., in detail. A genetically manipulated mouse line, which lacks 2001; Ikegami et al., 2007); ii) postnatal increase in level of PGs1 (Campbell et al., 2002), a component of the TTLL1- MAP1A in developing mouse brain (Schoenfeld et al., 1989); containing polyglutamylase complex (Regnard et al., 2003; iii) the important role of MAP1A in activity-dependent Janke et al., 2005), provides strong evidence. The PGs1- dendritic growth and branching (Szebenyi et al., 2005).

18 Unique Post-Translational Modifications in Specialized Microtubule Architecture

Fig. 4. Distribution of polyglutamylated and polyglycylated tubulins. Note that the subunits of tubulin that are well polyglutamylated are different between somato-dendrites and axons. Currently, no evidence is available that polyglycylated tubulin is detected in centrioles.

The roles of tubulin polyglutamylation in flagella or cilia Polyglycylation are still subject to controversy. The PGs1-deficient mouse shows malformation of sperm flagella, but not cilia in Polyglycylated tubulin is abundantly detected in axonemal trachea, nasal duct, or oviduct (Campbell et al., 2002), MTs (Redeker et al., 1994) (Fig. 4). In polyglycylation, a suggesting that tubulin polyglutamylation is required for poly-glycine chain is bound to a glutamate residue in tubu- axoneme construction and that the modification has distinct lin C-terminal region. In contrast to polyglutamylation, roles in flagella and cilia. Studies where modification- polyglycylation does not alter the net charge of the tubulin specific antibodies are used provide supportive evidence. C-terminal (Fig. 3). This simple addition of a mere poly- Ciliary motility of human respiratory epithelium is signifi- peptide that has no outstanding characteristic had made it cantly inhibited by injection of mAb GT335 (Million et al., difficult for researchers to anticipate the function of the 1999). A similar result is observed in a work where sea modification. However, the roles of tubulin polyglycylation urchin sperm is used. The flagellar motility was affected by are now beginning to be unveiled, as TTLLs 3, 8, and 10 injection of another mAb B3 into the sperm (Gagnon et al., are identified as polyglycylation-performing enzymes, the 1996). Although the numerous studies employing anti- polyglycylases (or glycine ligases) (Ikegami et al., 2008; bodies indicate the importance of tubulin polyglutamylation Ikegami and Setou, 2009; Rogowski et al., 2009; Wloga et in ciliary or flagellar motility, Tetrahymena, a ciliated pro- al., 2009b). tozoan swims normally even if it lacks both TTLL1 and While understanding the physiological roles of the tubu- TTLL9 (Wloga et al., 2008). The role of tubulin poly- lin polyglycylation in mammals has been slow to emerge, glutamylation may differ in organism species. To resolve elegant works using Tetrahymena have provided interesting these questions, we anticipate the development of mamma- evidence for the roles of tubulin polyglycylation. Mutations lian models, the TTLLs of which are genetically modified. in polyglycylation sites of β-tubulin, but not of α-tubulin, A recent work investigating zebrafish provides evidence have been reported to impair cellular motility and cytokine- for broad function of tubulin polyglutamylation in the cilia sis (Xia et al., 2000). Ultrastructurally, the axonemes of the of the whole body. A knockdown of Fleer, a newly identi- mutant lose the central-pair MTs and B-subfibers (Thazhath fied regulator of tubulin polyglutamylation, or TTLL6 et al., 2002), suggesting that tubulin polyglycylation has results in structural and functional abnormalities throughout a pivotal role in constructing ciliary axonemes. More the whole body (Pathak et al., 2007). However, the function recently, it has been revealed that tubulin polyglycylation by of the modification in centrioles is blurrier. The only evi- TTLL3 is essential for proper cilia assembly in Tetrahymena dence available is that mAb GT335 destabilizes the centri- and zebrafish (Wloga et al., 2009b). Interestingly, TTLL3- oles when injected into living cells (Bobinnec et al., 1998a). deficient Tetrahymena shows a counter-increase in poly- To achieve a more direct and accurate understanding glutamylation along with a decrease in tubulin polyglycyla- requires more robust investigations, e.g. those examining tion (Wloga et al., 2009b), which is consistent with the polyglutamylation-performing TTLL knockout animals. concept of cross-talk of PTMs in α- and β-tubulin C-termini (Redeker et al., 2005). The increase in polyglutamylation

19 K. Ikegami and M. Setou

Table I. ENZYMES AND FUNCTIONS OF UNIQUE TUBULIN PTMS

PTMs Enzymes Functions Detyrosination/tyrosination (CCP1/Nna1)a)/TTL · Regulating CLIP-170 binding and localization · Modulating kinesin-1 (KIF5) binding and trafficking · Regulating MT stability Polyglutamylation TTLL1, 4, 5, 6, 7, 9, 11, 13 · Modulating kinesin-3 (KIF1) binding and trafficking · Modulating MAP1-MTs interaction · Essential for sperm flagella formation · Important for neurite extension Polyglycylation TTLL3, 8, 10 · Acting as a suppressor of polyglutamylation · Essential for cilia formation a) The detyrosination-performing enzyme is still only a candidate. could explain the cause of abnormal cilia formation, as tier. It is amply clear that we are standing on the threshold over-polyglutamylation, manipulated by the overexpression of a vast field of studies on tubulin PTMs. of the polyglutamylase TTLL6, destabilizes axonemal microtubules (Wloga et al., 2009a). These findings can pro- Acknowledgements. This review was written as part of the Young Scien- vide a cue for understanding the molecular machinery tist Award for Best Presentation of the Japan Society for Cell Biology st whereby tubulin polyglycylation performs its function. The awarded to Koji Ikegami at the 61 JSCB Annual Meeting. The authors would like to express their heartfelt thanks to Prof. Kozo Kaibuchi, the MT-severing enzyme katanin might be one of the machiner- chair of the 61st JSCB Annual Meeting, and the organizing committee of ies since the phenotypes of the mutant MT are copied in the 61st JSCB Annual Meeting for giving us this opportunity to write this katanin-deficient organism (Sharma et al., 2007), though it review. We are also grateful to the present and former members of the remains unclear whether or not katanin recognizes polygly- Setou laboratory of the Hamamatsu University School of Medicine and the cylation or polyglutamylation. 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