Tyrosines in the kinesin-5 head domain are necessary for phosphorylation by Wee1 and for mitotic spindle integrity.
Mitotic spindle assembly and maintenance relies on kinesin-5 motors that act as bipolar homotetramers to crosslink microtubules. Kinesin-5 motors have been the subject of extensive structure-function analysis, but the regulation of their activity in the context of mitotic progression remains less well understood. We report here that Drosophila kinesin-5 (KLP61F) is regulated by Drosophila Wee1 (dWee1). Wee1 tyrosine kinases are known to regulate mitotic entry via inhibitory phosphorylation of Cdk1. Recently, we showed that dWee1 also plays a role in mitotic spindle positioning through gamma-tubulin and spindle fidelity through an unknown mechanism. Here, we investigated whether a KLP61F-dWee1 interaction could explain the latter role of dWee1. We found that dWee1 phosphorylates KLP61F in vitro on three tyrosines within the head domain, the catalytic region that mediates movement along microtubules. In vivo, KLP61F with tyrosine-->phenylalanine mutations fails to complement a klp61f mutant and dominantly induces spindle defects similar to ones seen in dwee1 mutants. We propose that phosphorylation of the KLP61F catalytic domain by dWee1 is important for the motor's function. This study identifies a second substrate for a Wee1 kinase and provides evidence for phosphoregulation of a kinesin in the head domain.
Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309-0347, USA.
Kristin
K
Garcia
Jason
J
Stumpff
Tod
T
Duncan
Tin Tin
TT
Su
D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology* / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D016547:Kinesins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D008941:Spindle Apparatus / Q000378:metabolism / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; C084034:Klp61F protein, Drosophila; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C508847:Wee1 protein, Drosophila; D016547:Kinesins