Polyglutamylation is a post-translational modification with a broad range of substrates

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins, and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.     

Identification of Tubulin Deglutamylase among Caenorhabditis elegans and Mammalian Cytosolic Carboxypeptidases (CCPs)

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.     

Phosphinic acid-based inhibitors of tubulin polyglutamylases

Tubulin is subject to a reversible post-translational modification involving polyglutamylation and deglutamylation of glutamate residues in its C-terminal tail. This process plays key roles in regulating the function of microtubule associated proteins, neuronal development, and metastatic progression. This study describes the synthesis and testing of three phosphinic acid-based inhibitors that have been designed to inhibit both the glutamylating and deglutamylating enzymes. The compounds were tested against the polyglutamylase TTLL7 using tail peptides as substrates (100 μM) and the most potent inhibitor displayed an IC₅₀ value of 150 μM. The incorporation of these compounds into tubulin C-terminal tail peptides may lead to more potent TTLL inhibitors.     

Post-translational modifications of tubulin in the nervous system

Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Δ2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.     

The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation

Cilia and basal bodies are essential organelles for a broad spectrum of functions, including the development of left-right asymmetry, kidney function, cerebrospinal fluid transport, generation of photoreceptor outer segments, and hedgehog signaling. Zebrafish fleer (flr) mutants exhibit kidney cysts, randomized left-right asymmetry, hydrocephalus, and rod outer segment defects, suggesting a pleiotropic defect in ciliogenesis. Positional cloning flr identified a tetratricopeptide repeat protein homologous to the Caenorhabditis elegans protein DYF1 that was highly expressed in ciliated cells. flr pronephric cilia were shortened and showed a reduced beat amplitude, and olfactory cilia were absent in mutants. flr cilia exhibited ultrastructural defects in microtubule B-tubules, similar to axonemes that lack tubulin posttranslational modifications (polyglutamylation or polyglycylation). flr cilia showed a dramatic reduction in cilia polyglutamylated tubulin, indicating that flr encodes a novel modulator of tubulin polyglutamylation. We also found that the C. elegans flr homologue, dyf-1, is also required for tubulin polyglutamylation in sensory neuron cilia. Knockdown of zebrafish Ttll6, a tubulin polyglutamylase, specifically eliminated tubulin polyglutamylation and cilia formation in olfactory placodes, similar to flr mutants. These results are the first in vivo evidence that tubulin polyglutamylation is required for vertebrate cilia motility and structure, and, when compromised, results in failed ciliogenesis.     

Distinct roles of α‐ and β‐tubulin polyglutamylation in controlling axonal transport and in neurodegeneration

Tubulin polyglutamylation is a post‐translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The “tubulin code” hypothesis predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation‐specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α‐tubulin, while TTLL7 modifies β‐tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis.     

Tubulin glycylases and glutamylases have distinct functions in stabilization and motility of ependymal cilia

Microtubules are subject to a variety of posttranslational modifications that potentially regulate cytoskeletal functions. Two modifications, glutamylation and glycylation, are highly enriched in the axonemes of most eukaryotes, and might therefore play particularly important roles in cilia and flagella. Here we systematically analyze the dynamics of glutamylation and glycylation in developing mouse ependymal cilia and the expression of the corresponding enzymes in the brain. By systematically screening enzymes of the TTLL family for specific functions in ependymal cilia, we demonstrate that the glycylating enzymes TTLL3 and TTLL8 were required for stability and maintenance of ependymal cilia, whereas the polyglutamylase TTLL6 was necessary for coordinated beating behavior. Our work provides evidence for a functional separation of glutamylating and glycylating enzymes in mammalian ependymal cilia. It further advances the elucidation of the functions of tubulin posttranslational modifications in motile cilia of the mammalian brain and their potential importance in brain development and disease.     

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.     

Coordination of posttranslational modifications of bovine brain alpha-tubulin. Polyglycylation of delta2 tubulin

Microtubules participate in a large number of intracellular events including cell division, intracellular transport and secretion, axonal transport, and maintenance of cell morphology. They are composed of tubulin, a heterodimeric protein, consisting of two similar polypeptides alpha and beta. In mammalian cells, both alpha- and beta-tubulin occur as seven to eight different genetic variants, which also undergo numerous posttranslational modifications that include tyrosination-detyrosination and deglutamylation, phosphorylation, acetylation, polyglutamylation, and polyglycylation. Tyrosination-detyrosination is one of the major posttranslational modifications in which the C-terminal tyrosine residue in alpha-tubulin is added or removed reversibly. Although this modification does not alter the assembly activity of tubulin in vitro, these two forms of tubulin have been found to be distributed differently in vivo and are also correlated with microtubule stability (Gunderson, G. G., Kalnoski, M. H., and Bulinski, J. C. (1984) Cell 38, 779-789). Thus, the question arises as to whether these two forms of tubulin differ in any other modifications. In an effort to answer this question, the tyrosinated and the nontyrosinated forms of the alpha1/2 isoform have been purified from brain tubulin by immunoaffinity chromatography. matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis of the C-terminal peptide revealed that the tyrosinated form is polyglutamylated with one to four Glu residues, while the Delta2 tubulin is polyglycylated with one to three Gly residues. These results indicate that posttranslational modifications of tubulin are correlated with each other and that polyglutamylation and polyglycylation of tubulin may have important roles in regulating microtubule assembly, stability, and function in vivo.     

Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A -related kinase 5

BACKGROUNDTubulins, building blocks of microtubules, are modified substrates of diverse post-translational modifications including phosphorylation, polyglycylation and polyglutamylation. Polyglutamylation of microtubules, catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family, can regulate interactions with molecular motors and other proteins. Due to the diversity and functional importance of microtubule modifications, strict control of the TTLL enzymes has been suggested.AIMTo characterize the interaction between never in mitosis gene A-related kinase 5 (NEK5) and TTLL4 proteins and the effects of TTLL4 phosphorylation.METHODSThe interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5 (a.a. 260–708) as bait and confirmed by immunoprecipitation. The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced.RESULTSHere, we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity. We further show that NEK5 can also interact with TTLL5 and TTLL7. The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4. The same effects were observed after the expression of the catalytically inactive form of NEK5. This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues.CONCLUSIONOur results demonstrate, for the first time, the regulation of TTLL activity through phosphorylation, pointing to NEK5 as a potential effector kinase. We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.     

A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures

CCDC39 and CCDC40 were first identified as causative mutations in primary ciliary dyskinesia patients; cilia from patients show disorganized microtubules, and they are missing both N-DRC and inner dynein arms proteins. In Chlamydomonas, we used immunoblots and microtubule sliding assays to show that mutants in CCDC40 (PF7) and CCDC39 (PF8) fail to assemble N-DRC, several inner dynein arms, tektin, and CCDC39. Enrichment screens for suppression of pf7; pf8 cells led to the isolation of five independent extragenic suppressors defined by four different mutations in a NIMA-related kinase, CNK11. These alleles partially rescue the flagellar length defect, but not the motility defect. The suppressor does not restore the missing N-DRC and inner dynein arm proteins. In addition, the cnk11 mutations partially suppress the short flagella phenotype of N-DRC and axonemal dynein mutants, but do not suppress the motility defects. The tpg1 mutation in TTLL9, a tubulin polyglutamylase, partially suppresses the length phenotype in the same axonemal dynein mutants. In contrast to cnk11, tpg1 does not suppress the short flagella phenotype of pf7. The polyglutamylated tubulin in the proximal region that remains in the tpg1 mutant is reduced further in the pf7; tpg1 double mutant by immunofluorescence. CCDC40, which is needed for docking multiple other axonemal complexes, is needed for tubulin polyglutamylation in the proximal end of the flagella. The CCDC39 and CCDC40 proteins are likely to be involved in recruiting another tubulin glutamylase(s) to the flagella. Another difference between cnk11-1 and tpg1 mutants is that cnk11-1 cells show a faster turnover rate of tubulin at the flagellar tip than in wild-type flagella and tpg1 flagella show a slower rate. The double mutant shows a turnover rate similar to tpg1, which suggests the faster turnover rate in cnk11-1 flagella requires polyglutamylation. Thus, we hypothesize that many short flagella mutants in Chlamydomonas have increased instability of axonemal microtubules. Both CNK11 and tubulin polyglutamylation play roles in regulating the stability of axonemal microtubules.     

Growth cones: the mechanism of neurite advance

Growth cones are the highly motile structures found at the tips of growing axons and dendrites (neurites), which extend from neurones, during the development of the nervous system. They function both as detectors and transducers of extrinsic guidance cues and as regions where the neurite assembly, advance cannot occur. Assembly of the neurite cytoskeleton in growing neurites chiefly involves microtubule assembly at the growth cone. Some of the factors that may influence microtubule assembly in growth cones are becoming apparent and include post-translational modification of tubulin itself and microtubule associated proteins, particularly tau and MAP1B.     

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.     

Class II tubulin, the major brain beta tubulin isotype is polyglutamylated on glutamic acid residue 435.

Protein sequencing shows that porcine brain tubulin retains the N-terminal sequences of alpha and beta tubulin after a mild treatment with subtilisin. C-terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of alpha tubulin on glutamic acid residue 445 reported by others and show in addition that class II beta tubulin, the major beta tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.     

A Highlights from MBoC Selection: A conserved flagella-associated protein in Chlamydomonas,FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9

Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase–like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility.     

Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic-neuron-like phenotype. Comparison of the phosphoprotein patterns of the cells by SDS PAGE after various times of NGF treatment revealed a high molecular weight (Mr greater than or approximately 300,000) band whose relative intensity progressively increased beyond 2 d of NGF exposure. This effect was blocked by inhibitors of RNA synthesis and did not require neurite outgrowth or substrate attachment. The enhancement by NGF occurred in serum-free medium and was not produced by exposure to epidermal growth factor, insulin, dibutyryl cAMP, or dexamethasone. Several different types of experiments indicated that this phosphoprotein corresponds to a high molecular weight (HMW) microtubule-associated protein (MAP). These included cross-reactivity with antiserum against brain HMW MAPs, co-cycling with microtubules and co-assembly with tubulin in the presence of taxol. The affected species also co-migrated in SDS PAGE gels with brain MAP1 and, unlike MAP2, precipitated upon boiling. Studies with [35S]-methionine-labeled PC12 cells indicated that at least a significant proportion of this effect of NGF was due to increased levels of protein rather than to mere enhancement of phosphorylation. On the basis of the apparent effects of MAPs on the formation and stabilization of microtubules and of the importance of microtubules in production and maintenance of neurites, it is proposed that induction of a HMW MAP may be one of the steps in the mechanism whereby NGF promotes neurite outgrowth. Furthermore, these findings may lead to an understanding of the role of MAP1 in the nervous system.     

Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates

Polyglutamylation is a widely distributed posttranslational modification of tubulin that can be demonstrated either by biochemical analysis or by the use of specific antibodies like GT335. Western blotting using GT335 demonstrated that polyglutamylated tubulin is enriched in isolated basal apparatus of Spermatozopsis similis. Single- and double-labeling experiments, using indirect immunofluorescence and immunogold electron microscopy of isolated cytoskeletons of S. similis and Chlamydomonas reinhardtii, revealed that polyglutamylated tubulin was predominately present in the basal bodies and the proximal part of the axonemes. Using immunogold labeling of whole mounts of Spermatozopsis cytoskeletons, we obtained evidence for a predominant occurrence of polyglutamylated tubulin in the B-tubule of the axonemal doublets. Polyglutamylation occurs early during premitotic basal body assembly in S. similis, whereas the probasal bodies of Chlamydomonas, which are present through interphase, showed a reduced staining with GT335 indicating that polyglutamylation is involved in basal body maturation. During flagella regeneration of C. reinhardtii, polyglutamylation preceded detyrosination and became visible shortly after the onset of flagellar regeneration. In C. reinhardtii and S. similis polyglutamylated tubulin was absent or highly reduced in the flagellar transition region, a specialized part of the flagellum linking the basal body to the axoneme. Furthermore, the transition region and the neighboring part of the axoneme showed reduced staining with L3, an antibody directed against detyrosinated tubulin. The results indicate that differences in the modification pattern can occur in a confined area of individual microtubules. The deficiency of polyglutamylated and detyrosinated tubulin in the transition region could have functional implications for flagellar turnover or excision.     

Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth

We have used the nerve growth factor (NGF)-responsive line of PC12 pheochromocytoma cells as a model system to study microtubule specializations associated with neurite outgrowth. PC12 cells treated with NGF cease proliferating and extend neurites. Long-term NGF treatment results in a two- to threefold increase in the proportion of total cellular tubulin that is polymerized in PC12 cells. The increase in this parameter first becomes apparent at 2-4 d with NGF and increases steadily thereafter. Several changes in microtubule-associated proteins (MAPs) of PC12 cells also occur after exposure to NGF. In immunoprecipitation assays, we observed the levels of MAP-2 to increase by at least several-fold after treatment with NGF. We also found that the compositions of three MAP classes with apparent Mr of 64K, 67K, and 80K are altered by NGF treatment. These MAPs, recently designated "chartins," are biochemically and immunologically distinct from the similarly-sized tau MAPs (Peng et al., 1985 Brain Res. 361: 200; Magendantz and Solomon, 1985 Proc. Natl. Acad. Sci. 82: 6581). In two-dimensional isoelectric focusing x SDS polyacrylamide gels, each chartin MAP class resolves into a set of proteins of similar apparent Mr but distinct pI. Peptide mapping analyses confirm that the isoelectric variants comprising each chartin MAP class are closely related in primary structure. Several striking differences in the composition of the chartin MAPs of PC12 cells grown with or without NGF were consistently observed. In particular, following longterm NGF treatment, the abundances of the more acidic variants of each chartin MAP class were markedly enhanced relative to the more basic members. This occurs without substantial changes in the abundance of each MAP class as a whole relative to total cell protein. The combined results of in vivo phosphorylation and peptide mapping experiments indicate that the NGF-inducible chartin MAP species are not primary translation products, but are generated posttranslationally, apparently by differential phosphorylation of other chartin MAPs. These observations suggest that NGF treatment of PC12 cells leads to changes in the posttranslational processing of the chartin MAPs. The time course of these changes closely resembles that for the increase in the proportion of cellular tubulin that is polymerized and for neurite outgrowth. One of the important events in the growth and stabilization of neurites appears to be the formation of microtubule bundles that extend from the cell body to the tips of the neurites.(ABSTRACT TRUNCATED AT 400 WORDS)