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...

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.

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