Cell Motility and the Cytoskeleton 46:1–5 (2000) Views and Reviews Phosphorylation of Tubulin Tyrosine Ligase: A Potential Mechanism for Regulation of ␣-Tubulin Tyrosination Haitham T. Idriss Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife, Scotland, United Kingdom and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas, USA The tubulin tyrosination/detyrosination cycle is a well-established posttranslational modification, which is carried out by two enzymes: Tubulin Tyrosine Ligase (TTL) and Tubulin Tyrosine Carboxypeptidase (TTCP). In this paper, I present evidence suggesting that the cycle itself is under the hierarchical control of reversible phosphorylation and that PKC mediated phosphorylation of TTL inhibits its activity, thereby preventing tubulin tyrosination. Phosphorylation of TTL is predicted to occur in a postulated Mg⫹⫹/-ATP binding fold, leading to inhibition of Mg⫹⫹/ATP binding and TTL mediated catalysis. The implications of such control are also discussed. Cell Motil. Cytoskeleton 46:1–5, 2000. © 2000 Wiley-Liss, Inc. Key words: tubulin; microtubules; tubulin tyrosine carboxypeptidase; tubulin tyrosine ligase; PKC INTRODUCTION Microtubules are a major component of the cytoskeleton, which are involved in a diverse range of cell functions that include maintenance of cell shape and integrity and formation of the microtubule spindle that separates chromosomes during cell division and intracellular transport [Brinkley, 1997; Rieder and Khodjakov, 1997; Sheetz, 1999]. Tubulin, the protein building block of microtubules, is a heterodimer that is subject to a number of covalent posttranslational modification on its ␣- and/or ␤-subunits, such as phosphorylation, acetylation, and tyrosination. The ␣-tubulin tyrosination/detyrosination cycle is a posttranslational modification that is unique to tubulin and may be important for certain microtubule functions. The modification involves the removal of a genetically encoded tyrosine residue from the C-terminus of ␣-tubulin, by the enzyme tubulin tyrosine carboxypeptidase (TTCP), and the subsequent re-addition of tyrosine by another enzyme, termed tubulin tyrosine ligase (TTL). The modification may be important © 2000 Wiley-Liss, Inc. for many cellular processes [for review see Idriss, 2000a; Barra et al., 1988]. An important question is what controls tubulin tyrosination/detyrosination? Here I argue that reversible phosphorylation is a hierarchical control for the cycle of tubulin tyrosination. TTL HAS SEVERAL POTENTIAL PHOSPHORYLATION SITES TTL is a 40 kDa enzyme that catalyses the addition of tyrosine to the C-terminus of ␣-tubulin isoforms that terminate with the residues Gly-Glu-Glu (GEE), in a reaction that hydrolyses ATP and requires Mg⫹⫹ [reviewed in Idriss, 2000a]. The enzyme was recently cloned Contract grant sponsor: Hoechst Marion Roussel (France). *Correspondence to: H.T. Idriss, School of Biomedical Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, Scotland, UK. E-mail: email@example.com. Received 12 January 2000; accepted 27 January 2000 2 Idriss Fig. 1. Primary sequence of tubulin tyrosine ligase (TTL). Conserved amino acids within ATP-grasp enzymes that are involved in ATP binding are in white fonts and shaded in black [from Galperin and Koonin, 1997] or shaded dark grey [from Dideberg and Bertrand, 1998]. Other conserved amino acids are in black font and shaded light grey [from Galperin and Koonin, 1997]. Numbers indicate sequences lying within a phosphorylation consensus sequence, which are underlined. Potential phosphate-acceptor residues are in italics. Although both Serine-153 and Serine-226 are conserved, only one complete PKC consensus sequence, K/RXS, is conserved throughout the ATPgrasp enzyme family (150KSS153 for TTL). from porcine brain by Ersfeld et al. and the authors reported the presence of the consensus phophorylation site, 73RKAS76, to PKA [Ersfeld et al., 1993]. However, close inspection of the TTL sequence (Fig. 1) reveals the presence of at least eight-consensus phosphorylation sites to PKA, PKC, CKII, or Tyrosine kinase (Table I). The majority of these sites were to PKC/PKA and the Tyrosine kinase consensus site was also present within a PKA phosphorylation consensus sequence. The prevalence of such sites suggests that TTL, itself, and therefore tubulin tyrosination, could be regulated by reversible phosphorylation in cells. This is further supported by published data from several studies presented below. containing molecule to an amino or thiol-containing molecule. Similarly, Dideberg and Bertrand  also proposed that TTL has a shared fold with the Gultathione synthetase (GTHase) ADP-forming family. The conserved amino acids within ATP-grasp motifs that interact with Mg⫹⫹/ATP and which are important for catalysis are shown for TTL in Figure 1. Several of these amino acids are part of or lie close to a phosphorylation consensus sequence to PKC (e.g., 150KSS153) and could be affected by the addition of the negative charge of a phosphate moiety. This makes phosphorylation a likely mechanism for regulating TTL’s activity by interfering with, for example, ATP binding. The crystal structure of TTL, once determined, should give good insight into how important structural motifs could be regulated by covalent modifications. TTL IS AN ATP-GRASP ENZYME Using the PSI-BLAST method for sequence database analysis, Galperin and Koonin  classified TTL as an ATP-grasp enzyme, based on the presence within TTL’s primary sequence, of an ATP-grasp (or palmate) fold that is unique to a family of nucleotide triphosphate-binding enzymes. The ATP-grasp enzymes catalyse the ATP-dependent ligation of a carboxylate- TABLE I. Potential Phosphorylation Sites Within TTL* Target site 1 2 3 4 5 6 7 8 Peptide 73 76 RKAS KTS84 123 SRTD126 150 KSS153 223 RTAS226 248 KEYS251 248 KEYS251 304 STR306 82 Kinase Target residue PKA/PKC PKC CKII PKC PKA/PKC Tyr-Kinase PKA/PKC PKC S76 S84 T125 S153 S226 Y250 S251 S304 *Phosphorylation consensus sequences are based on those described by Pearson and Kemp : CKII sites, (S/T XX E/DX); PKA sites, (K/R XX S/T); PKC sites, (S/T X R/K, R/K X S/T, K/R XX S/T); Tyr-kinase sites, (X E/D Y X). HIERARCHICAL CONTROL OF THE TYROSINATION CYCLE The tyrosination cycle is regulated by microtubule assembly/disassembly since TTCP acts preferentially on assembled microtubules, whilst TTL utilises free tubulin dimer for the tyrosination reaction [MacRae, 1997]. Additionally, tyrosination levels are limited by the presence of a non-tyrosinatable tubulin subfraction, termed ⌬2 tubulin [Paturle-Lafanechere et al., 1991]. However, it is possible hierarchical control is conferred on the tyrosination cycle through posttranslational modification(s), in particular through reversible phosphorylation of TTL. The abundance of phosphorylation consensus sequences within TTL (Table I) and work on the macrophage microtubule network, support this suggestion. Macrophages possess highly dynamic microtubules (t1/2 ⬇ 30 sec) that are predominantly tyrosinated. Robinson and Vandre  demonstrated that exposure to phorbol esters (activators of PKC) caused complete loss of tubulin tyrosination, which was restored upon removal of the phorbol Inhibition of ␣-Tubulin Tyrosination esters. This loss of tyrosinated tubulin may have been due to enhancement of TTCP activity. However, such activation of TTCP is unlikely, as TTCP interaction with microtubules is hampered by polycations/polyanions, which lead to downregulation of detyrosination in vitro [Lopez et al., 1990; Webster and Oxford, 1996]. The same downregulation would be anticipated should TTCP become phosphorylated. Indeed, Sironi et al  reported that conditions favouring phosphorylation lead to diminished TTCP activity on microtubules and that such downregulation could be prevented through the presence of protein phosphatases. Hence, phosphorylation of TTCP is expected to lead to sustained tyrosination of tubulin rather than complete detyrosination. The effect of phosphorylation on TTCP, which is a microtubule interactive protein, is therefore similar to what is observed for MAPs whereby phosphorylation of MAPs (e.g., MAP-2) induces diminished interaction with microtubule walls [Avila et al., 1994]. Since TTCP phosphorylation is an unlikely explanation for the observed, phorbol ester-induced, total loss of microtubule tyrosination in microphages, two possibilities remain: that PKC mediated phosphorylation of either tubulin or TTL is responsible for such a phenomenon. TTL has a binding site on ␤-tubulin [Wehland and Weber, 1987] and phosphorylation of ␤-tubulin may cause diminished TTL binding to tubulin, thereby preventing tyrosination of the tubulin dimer. Likewise, phosphorylation of ␣-tubulin may prevent TTL mediated incorporation of the tyrosine residue onto the C-terminus of this subunit. However, the complete loss of tyrosination in macrophages would require phosphorylation of all the ␤-tubulin isoforms or the tyrosinatable ␣-tubulin isoforms, which is unlikely since only one tubulin isoform, ␤III, is known to be phosphorylated in vivo [Ludueña, 1998]. Further, there is no indication that PKC utilises tubulin as a substrate in vivo, although several other kinases have been shown to phosphorylate tubulin [MacRae, 1997]. PHOSPHORYLATION OF TTL MAY INTERFERE WITH ATP BINDING The simplest explanation for the above observations in macrophages would be that PKC-mediated phosphorylation of TTL inhibits its catalytic activity. Although, such phosphorylation may occur on any of the potential sites present in TTL, it is likely this occurs on serine-153 within the PKC consensus sequence 151 KSS153, preventing Mg⫹⫹/ATP binding. Residues in the aforementioned sequence are conserved among several ATP-grasp enzyme protein families [Galperin and Koonin, 1997], while lysine-151 is part of the Mg⫹⫹/ ATP binding fold and is conserved in GTHase ADP- 3 forming synthetases [Dideberg and Bertrand, 1998]. Phosphorylation introduces a negatively charged moiety on serine-153 and this may well interfere with binding of ATP to TTL, preventing catalysis. Conservation of serine-153 suggests that phosphorylation may be a common mechanism for regulating the activities of ATPgrasp enzymes. Interestingly, Robinson and Vandre observed that in phorbol ester–treated macrophages, microtubules that had recovered from nocodazole-induced disassembly were first observed to be tyrosinated. Therefore, it seems that somehow nocodazole prevented the presumed PKC mediated inhibition of tubulin tyrosination. One explanation could be that the introduction of nocodazole into cells activates cellular phosphatases or kinases, thereby causing dephosphorylation or preventing phosphorylation of TTL. This, in turn, maintains TTL in an active form. Microtubule depolymerising drugs, such as nocodazole, have several effects on cellular processes involving signal transduction and the presence of nocodazole may induce the activity of a membrane transporter(s) such as the membrane transporter, P-glycoprotein, which is responsible for multi-drug resistance in cancer cells, by activating protein kinases/phosphatases. Reversible phosphorylation may regulate P-glycoprotein activities [reviewed in Idriss et al., 2000] and it is possible that the presence of nocodazole indirectly causes TTL phosphorylation during initiation of molecular efflux. In this respect, the presence of nocodazole may have balanced out the level of TTL phosphorylation in macrophages, leading to restoration of tubulin tyrosination. Additional support for a role for phosphorylation in regulating tubulin tyrosination comes from studies in fibroblasts, which only have a small population of detyrosinated microtubules. In these cells, lysophosphatidic acid–induced cell signalling leads to stimulation of tubulin detyrosination and this seems to be mediated by the small GTPase, Rho [Gundersen and Cook, 1999]. Such regulation of detyrosinated microtubule formation could occur by regulating the activities of TTL/TTCP through reversible phosphorylation. Interestingly, a number of serine/threonine protein kinases are activated by the small GTPase, Rho [Narumiya, 1996). IMPLICATIONS OF TTL PHOSPHORYLATION The level of tubulin tyrosination may be controlled through regulating the assembled/free tubulin fraction, TTL/TTCP activities and/or the expression of the nontyrosinatable (⌬2) tubulin isoform. However, regulating TTL/TTCP activities confers a finer control of the tyrosination cycle and would make this cycle sensitive to fluctuations in kinase/phosphatase activities, which vary during cell cycling and differentiation. Together with the 4 Idriss Fig. 2. Schematic representation of tubulin nitrotyrosination. Elimination of “abnormal” cells is postulated to occur due to ␣-tubulin (␣-Tu.GEE) nitrtyrosination-mediated microtubule dysfunction, which possibly affects the binding of MAPs/motors to microtubules. Only tubulin with a C-terminus GEE residues is utilised as a substrate by TTL. TTCP may not function to remove the incorporated nitrotyrosine (NO2Y). TNF ␣ may enhance tubulin nitrotyrosination. Suppression of TTL activity may occur due to phosphorylation and this prevents tubulin nitrotyrosination, resulting in “abnormal” cells escaping apoptosis. Protein phosphatases (PP) may restore TTL activity. A marker for cell “abnormality” is an increase in nitrotyrosine levels, which is observed in many pathological situations. observation that phosphorylation may regulate the association/activity of TTCP [Sironi et al., 1997], reversible phosphorylation could confer hierarchical regulation of the tubulin tyrosination cycle independent of microtubule assembly/disassembly. Regulating TTL activity through PKC mediated phosphorylation may prove an interesting observation particularly since suppressed levels of TTL were associated with tumour progression [Lafanechere et al., 1998]. Inactivation of TTL through PKC-mediated phosphorylation may be a mechanism through which such suppression occurs in response to extracellular signalling, contributing towards tumourigenesis. PKC activity is known to increase during malignancy and phorbol esters are known tumour promoters. Likewise, elevation of PKA activity (e.g., due to increased c-AMP levels) may also lead to inhibition of TTL activity. It is not clear how TTL suppression contributes to tumourigenesis. However, recently it has been reported that TTL incorporates nitro- tyrosine onto tubulin in vivo, causing cellular dysfunction and apoptosis [Eiserich et al., 1999]. Since the levels of nitrotyrosine are elevated in a number of cancers [Ahn et al., 1999; Goto et al., 1999; Kojima et al., 1999], I suggest that tubulin nitrotyrosination may be a defence mechanism for eliminating “abnormal” cells, such as malignant ones. Suppression of TTL activity during malignancy would therefore result in bypass of tubulin nitrotyrosination, preventing elimination of cancer cells and contributing towards tumourigenesis (Fig. 2). Additionally, tumour necrosis factor ␣ (TNF␣)-insensitive tumour cells may be so because they also escape ␣-tubulin nitrotyrosination through phosphorylation and, therefore, inaction of TTL [Idriss, 2000b]. TTL may also turn out to be suppressed in other “abnormal” cells such as infected ones. The concept of eliminating potentially harmful cells is well established in, for example, the immune system [Abbas, 1996]. Inhibition of ␣-Tubulin Tyrosination CONCLUDING REMARKS Utilisation of TTL as a substrate for PKC and other kinase(s) in vitro should confirm the effect(s) such phosphorylation has on TTL activity. Amino acid sequencing and/or point mutation of the potential serine/threonine targets (e.g., to alanine or aspartic acid) should also confirm the site for phosphorylation and its consequence(s). Protein phosphatases (e.g., PP1 or PP2) can be tested for their ability to act on phosphorylated TTL. Confirmation that this is a general phenomenon can be obtained through testing the effects of phorbol esters on tubulin tyrosination in other cell types. 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