The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule-or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates -tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of -tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-3-tubulin S172D/E mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to -tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis. INTRODUCTIONIn eukaryotic cycling cells, interphase microtubules form a dynamic network that is essential for cell polarity and intracellular traffic. When cells enter into mitosis, the interphase microtubule network rearranges into a mitotic spindle that is responsible for faithful chromosome segregation between daughter cells. Microtubule dynamics and turnover increase strikingly as cells progress from interphase to mitosis, with a microtubule half-life of 5-10 min in interphase and of 60 -90 s in mitosis (Wittmann et al., 2001).Microtubule dynamics in cells rely in part on the intrinsic properties of the microtubule building block, the ␣--tubulin dimer, and its ability to bind and hydrolyze a GTP nucleotide (Mitchison and Kirschner, 1984). The tubulin dimer is subject to special posttranslational modifications such as glutamylation, tyrosination, and acetylation (MacRae, 1997;Westermann and Weber, 2003). There is little evidence for a role of these modifications in the regulation of microtubule dynamics. Tubulin can also be phosphorylated by several kinases (Westermann and Weber, 2003). However, tubulin phosphorylation has not been connected with the cell-cycle-dependent regulation of microtubule dynamics.Microtubule dynamics are also regulated by a number of microtubule effectors, including microtubule-associated proteins (MAPs), molecular motors such as kinesins, the Raslike GTPase Ran-GTP, microtubule plus end-directed proteins and tubulin-binding proteins (Andersen, 1999(Andersen, , 2000Carazo-Salas et al., 2001;Cassimeris, 2002;Heald and Nogales, 2002;Kinoshita et al., 2002;Galjart and Perez, 2003). These microtubule-or tubulin-associated proteins are themselves under the control of a...
Peptide deformylase (PDF) inhibitors have a strong potential to be used as a new class of antibiotics. However, recent studies have shown that the mitochondria of most eukaryotes, including humans, contain an essential PDF, PDF1A. The crystal structure of the Arabidopsis thaliana PDF1A (AtPDF1A), considered representative of PDF1As in general, has been determined. This structure displays several similarities to that of known bacterial PDFs. AtPDF1A behaves as a dimer, with the C-terminal residues responsible for linking the two subunits. This arrangement is similar to that of Leptospira interrogans PDF, the only other dimeric PDF identified to date. AtPDF1A is the first PDF for which zinc has been identified as the catalytic ion. However, the zinc binding pocket does not differ from the binding pockets of PDFs with iron rather than zinc. The crystal structure of AtPDF1A in complex with a substrate analog revealed that the substrate binding pocket of PDF1A displays strong modifications. The S1 binding pocket is significantly narrower, due to the creation of a floor from residues present in all PDF1As but not in bacterial PDFs. A true S3 pocket is created by the residues of a helical CD-loop, which is very long in PDF1As. Finally, these modified substrate binding pockets modify the position of the substrate in the active site. These differences provide guidelines for the design of bacterial PDF inhibitors that will not target mitochondrial PDFs.
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