CEA Direction des sciences du vivant - iRTSV : The tubulin tyrosination cycle

The tubulin tyrosination cycle

The tubulin tyrosination cycle

Tubulin is subject to specific post-translational modifications that principally affect the C-terminus of the α subunit. One of these modifications known as the tyrosination cycle involves the cyclic removal of the C-terminal tyrosine residue of the α-tubulin chain by an un-characterized tubulin carboxypeptidase (TCP) and the addition of a tyrosine residue at the same site by tubulin-tyrosine-ligase (TTL).

The tyrosination cycle has been discovered decades ago and shown to be highly conserved among eukaryotes. Yet, its significance has been elusive. However, over the past few years, observations from our laboratory have revealed dramatic effects of tyrosination cycle inhibition.

Tubulin tyrosine ligase (TTL)

A first key observation was that TTL behaves as a potential tumour suppressor. We have found both in animal models and in human tumors that TTL is frequently suppressed during tumor progression, the resulting tubulin detyrosination being associated with increased tumor aggressiveness [Lafanechère et al., 1998, Mialhe et al., 2001]. These observations indicate a strong selective advantage of TTL suppression and tubulin detyrosination for malignant cell proliferation and suggest that detyrosination may somehow affect mitosis integrity.
In most eukaryotic cells, the C-terminal amino acid of α-tubulin is aromatic: Tyr in mammals and Phe in Saccharomyces cerevisiae. We have developed S. cerevisiae strains expressing only Glu tubulin (Glu strain) and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends [Badin-Larcon et al., 2004] (PhD thesis of Anne-Claire Badin 2000-2003, directed by Didier Job).

Anne-Claire Badin-Larçon.
Rôle de l'acide-aminé C-terminal de la tubuline α au cours du cycle cellulaire chez la levure S. cerevisiae.
[Abstract]

In parallel, we have developed TTL deficient mice. TTL KO mice develop normally but die shortly after birth due to major disorganization of neuronal networks, including a disruption of the thalamo-cortical loop [Erck et al., 2005]. These defects correlate with anomalies affecting the morphogenesis of cultured TTL null neurons, which display an accelerated and erratic time course of neurite outgrowth and a premature axonal differentiation. Interestingly, these defects relate to a mis-localization of CLIP170, which is lacking in the growth cones of TTL null neurons [Erck et al., 2005].
We are currently investigating the molecular interaction of Glu tubulin with MTs + ends proteins such as CLIP170 proteins and EB1, in primary fibroblasts (MEFs). As expected from yeast and neuronal studies we find a defect in the interaction of Glu tubulin and CLIP170. Recent results seem to indicate that the mis-interaction between Glu MTs and Clip 170 can be extended to all MTs + ends proteins containing a CAP-Gly domain (CLIP 115, P150Glued).
Altogether, yeast and mice results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex.

The Tubulin Carboxy Peptidase (TCP)

Finally, we intend to identify and characterize the TCP protein. Several groups have tried, using biochemical techniques, to identify TCP without success. We plan to use several other approaches. The team of Laurence Lafanechère (CEA Grenoble) is searching for TCP inhibitors using High Throughout Screening (HTS). The identification of specific inhibitors should allow the purification of the TCP and its identification by mass spectrometry. We also want to use the human genome information to list all the carboxy peptidases and try to determine the features specific for the degradation of aromatic residue such as Tyr or Phe. This search should result in less than 50 candidates. We then, might use siRNA techniques to knock down all the candidates. After transfection of the siRNA in fibroblasts, we will evaluate the accumulation of Glu tubulin. If we are successful in the identification of the TCP, we will perform biochemical and cellular studies and do the gene invalidation of the TCP in mice.

References

Marcos S, Moreau J, Backer S, Job D, Andrieux A and Bloch-Gallego E
Tubulin tyrosination is required for the proper organization and pathfinding of the growth cone.
PLoS ONE, 2009, 4(4): e5405

Peris L, Wagenbach M, Lafanechère L, Brocard J, Moore AT, Kozielski F, Job D, Wordeman L and Andrieux A
Motor-dependent microtubule disassembly driven by tubulin tyrosination.
Journal of Cellular Biology, 2009, 185(7): 1159-1166

Erck C, Peris L, Andrieux A, Meissirel C, Gruber AD, Vernet M, Schweitzer A, Saoudi Y, Pointu H, Salin PA, Job D and Wehland J
A vital role of tubulin-tyrosine-ligase for neuronal organization.
Proceedings of the National Academy of Science USA, 2005, 102(22), 7853-7858

Badin-Larcon AC, Boscheron C, Soleilhac JM, Piel M, Mann C, Denarier E, Fourest Lieuvin A, Lafanechère L, Bornens M and Job D
Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin.
Proceedings of the National Academy of Science USA, 2004, 101(15): 5577-5582

Mialhe A, Lafanechere L, Treilleux I, Peloux N, Dumontet C, Bremond A, Panh MH, Payan R, Wehland J, Margolis RL and Job D
Tubulin detyrosination is a frequent occurrence in breast cancers of poor prognosis.
Cancer Research, 2001, 61(13): 5024-5027

Lafanechere L, Courtay-Cahen C, Kawakami T, Jacrot M, Rudiger M, Wehland J, Job D and Margolis RL
Suppression of tubulin tyrosine ligase during tumor growth.
Journal of Cell Science, 1998, 111( Pt 2): 171-81

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The tubulin tyrosination cycle