Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1.
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 beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-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-beta3-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 beta-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.
Fourest-Lieuvin
Anne
A
Laboratoire du Cytosquelette, INSERM Unité 366, CEA, 38054 Grenoble Cedex 9, France. anne.fourest-lieuvin@cea.fr
Peris
Leticia
L
Gache
Vincent
V
Garcia-Saez
Isabel
I
Juillan-Binard
Céline
C
Lantez
Violaine
V
Job
Didier
D
D000595:Amino Acid Sequence; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002417:Cattle; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012694:Serine / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D045163:Antibodies, Phospho-Specific; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D012694:Serine; D016203:CDC2 Protein Kinase