Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles (original) (raw)
Figure 1
Phosphorylation of Xenopus laevis Eg5 by Cdk1/cyclin B in vitro does not affect its velocity as measured in microtubule gliding assays.
(A) Schematic representation of the Eg5 sequence with the phosphorylation site for Cdk1 in the tail (threonine 937). (B) In vitro phosphorylation of wild-type Eg5 and Eg5T937A with [γ32P]ATP in buffer in the presence (+) or absence (−) of Cdk1/cyclin B (top). Autoradiography (32P) and coomassie-stained polyacrylamide gel (C) are shown. Quantitative measurement of radioactivity of the corresponding bands from the SDS-gel. The degree of phosphorylation for wild-type Eg5 is 87.9% and is strongly reduced to 4.0% for Eg5T937A. (C) Phosphorylation does not affect the velocity of Eg5 as measured in microtubule gliding assays: Histograms of gliding velocities of individual microtubules (top) and bar plot of averaged gliding velocities (bottom) produced by wild-type Eg5 (WT) and a non-phosphorylatable mutant (Eg5T937A) treated with or without Cdk1 kinase. Red lines are a Gauss fit, error bars are standard errors. The velocities of phosphorylated and unphosphorylated wild-type Eg5 (WT) are not statistically significantly different (p = 0.067, significance level 0.05); the 12% difference between the velocities of wild-type Eg5 (WT) and the non-phosphorylatable mutant (T937A) is statistically significant (p = 8.6×10−7, significance level 0.05).