вход по аккаунту



код для вставкиСкачать
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
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 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:
Received 12 January 2000; accepted 27 January 2000
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 [1998] 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.
Using the PSI-BLAST method for sequence database analysis, Galperin and Koonin [1997] 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
Target residue
*Phosphorylation consensus sequences are based on those described
by Pearson and Kemp [1991]: 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).
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
[1995] 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 [1997] 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].
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
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-
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).
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
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
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
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.
The possible involvement of reversible phosphorylation in regulating tubulin tyrosination suggests this
cycle may be more complex than initially thought and
that it may be subject to regulation by various extracellular stimuli as well as cell cycling. Microtubules play an
important role in signal transduction pathways and many
signalling molecules, including protein kinases and phosphatases, are associated with microtubules or microtubule binding proteins [Gundersen and Cook, 1999]. This
may also point to a potential function of this cycle in
pathological conditions.
I thank Drs. Dale Vandre for comments on his
work, Michael Galperin for the reprint, Martin Ryan for
his comments on the manuscript, and Jim Naismith for
support. I also thank Hoechst Marion Roussel (France)
for financial support.
Abbas AK. 1996. Die and let live: eliminating dangerous lymphocytes.
Cell 84:655– 657.
Ahn B, Han BS, Kim DJ, Ohshima H. 1999. Immunohistochemical
localization of inducible nitric oxide synthase and 3-nitrotyrosine in rat liver tumors induced by N-nitrosodiethylamine.
Carcinogenesis 20:1337–1344.
Avila J, Dominguez J, Diaz-Nido J. 1994. Regulation of microtubule
dynamics by microtubule-associated protein expression and
phosphorylation during neuronal development. Int J Dev Biol
Barra HS, Arce CA, Argarana CE. 1988. Posttranslational tyrosination/detyrosination of tubulin. Mol Neurobiol 2:133–53.
Brinkley W. 1997. Microtubules: a brief historical perspective. J Struct
Biol 118:84 – 6.
Dideberg O, Bertrand J. 1998. Tubulin tyrosine ligase: a shared fold
with the glutathione synthetase ADP-forming family. Trends
Biochem Sci 23:57– 8.
Eiserich JP, Estevez AG, Bamberg TV, Ye YZ, Chumley PH, Beckman JS, Freeman BA. 1999. Microtubule dysfunction by posttranslational nitrotyrosination of alpha- tubulin: a nitric oxidedependent mechanism of cellular injury. Proc Natl Acad Sci
USA 96:6365–70.
Ersfeld K, Wehland J, Plessmann U, Dodemont H, Gerke V, Weber K.
1993. Characterization of the tubulin-tyrosine ligase. J Cell
Biol 120:725–32.
Galperin MY, Koonin EV. 1997. A diverse superfamily of enzymes
with ATP-dependent carboxylate-amine/thiol ligase activity.
Protein Sci 6:2639 – 43.
Goto T, Haruma K, Kitadai Y, Ito M, Yoshihara M, Sumii K, Hayakawa N, Kajiyama G. 1999. Enhanced expression of inducible
nitric oxide synthase and nitrotyrosine in gastric mucosa of
gastric cancer patients [in process citation]. Clin Cancer Res
Gundersen GG, Cook TA. 1999. Microtubules and signal transduction.
Curr Opin Cell Biol 11:81–94.
Idriss HT. 2000a. Man to Trypanosome: the tubulin tyrosination/
detyrosination cycle revisited. Cell Motil Cytoskeleton 45:173–
Idriss HT. 2000b. Do TNF␣-insensitive cancer cells escape ␣-tubulin
nitrotyrosination? Nitric Oxide 4:1–3.
Idriss HT, Hannun YA, Boulpaep E, Basavappa S. 2000. P-glycoprotein regulation of volume activated Cl-channels: phosphorylation has the final say! J Physiol 524:629 – 636.
Kojima M, Morisaki T, Tsukahara Y, Uchiyama A, Matsunari Y, Mibu
R, Tanaka M. 1999. Nitric oxide synthase expression and nitric
oxide production in human colon carcinoma tissue. J Surg
Oncol 70:222–9.
Lafanechere L, Courtay-Cahen C, Kawakami T, Jacrot M, Rudiger M,
Wehland J, Job D, Margolis RL. 1998. Suppression of tubulin
tyrosine ligase during tumor growth. J Cell Sci 111:171– 81.
Lopez RA, Arce CA, Barra HS. 1990. Effect of polyanions and
polycations on detyrosination of tubulin and microtubules at
steady state. Biochim Biophys Acta 1039:209 –17.
Ludueña RF. 1998. Multiple forms of tubulin: different gene products
and covalent modifications. Int Rev Cytol 178:207–75.
MacRae TH. 1997. Tubulin post-translational modifications: enzymes
and their mechanisms of action. Eur J Biochem 244:265–78.
Narumiya S. 1996. The small GTPase Rho: cellular functions and
signal transduction. J Biochem (Tokyo) 120:215–28.
Pearson RB, Kemp BE. 1991. Protein kinase phosphorylation site
sequences and consensus specificity motifs: tabulations. Meth
in Enzymol 200:62– 81.
Paturle-Lafanechere L, Edde B, Denoulet P, Van Dorsselaer A, Mazarguil H, Le Caer JP, Wehland J, Job D. 1991. Characterization
of a major brain tubulin variant which cannot be tyrosinated.
Biochemistry 30:10523– 8.
Rieder CL, Khodjakov A. 1997. Mitosis and checkpoints that control
progression through mitosis in vertebrate somatic cells. Prog
Cell Cycle Res 3:301–12.
Robinson JM, Vandre DD. 1995. Stimulus-dependent alterations in
macrophage microtubules: increased tubulin polymerization
and detyrosination. J Cell Sci 108:645–55.
Sheetz MP. 1999. Motor and cargo interactions. Eur J Biochem 262:
19 –25.
Sironi JJ, Barra HS, Arce CA. 1997. The association of tubulin
carboxypeptidase activity with microtubules in brain extracts is
modulated by phosphorylation/dephosphorylation processes.
Mol Cell Biochem 170:9 –16.
Webster DR, Oxford MG. 1996. Regulation of cytoplasmic tubulin
carboxypeptidase activity in vitro by cations and sulfhydrylmodifying compounds. J Cell Biochem 60:424 –36.
Wehland J, Weber K. 1987. Tubulin-tyrosine ligase has a binding site
on beta-tubulin: a two-domain structure of the enzyme. J Cell
Biol 104:1059 – 67.
Без категории
Размер файла
77 Кб
Пожаловаться на содержимое документа