Figure 2. Phosphorylation of Eg5 by Cdk1 increases Eg5 binding to microtubules in buffer.(A) Schematic representation of the Eg5-GFP sequence. (B) In vitro phosphorylation of wild-type Eg5-GFP and Eg5T937A-GFP with [γ32P]ATP in buffer in the presence (+) or absence (−) of Cdk1/cyclin B. 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 of Eg5 wild-type is 75.6% and is strongly reduced to 5.9% for Eg5T937A-GFP. (C) Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro: Representative examples for time-averaged images of the Eg5-GFP intensity on single immobilized microtubules as measured by TIRF microscopy (top). Wild-type Eg5-GFP (WT-GFP) and the non-phosphorylatable mutant (T937A-GFP) treated with ATP in the presence or absence of Cdk1/cyclin B were added to immobilized microtubules at a concentration of 15 nM. Scale bar is 1 µm. Bar plot of average Eg5-GFP signals (bottom) as obtained from 45–90 microtubules per condition as described above. Error bars are standard errors. Insert: Loading control showing the amount of Eg5-GFP in the phosphorylation reactions used in the experiment. (D) Average intensity signals (left) and intensity ratios (right) of Eg5-GFP and Eg5T937A-GFP on microtubules after Cdk1/cyclin B treatment plotted as a function of different Eg5 construct concentrations. For the average intensity signals (left), the values (in A.U.) are for WT and T937A at the concentration of 1 nM: 109±7 and 11±2, at the concentration of 5 nM: 439±17 and 40±3, at the concentration of 15 nM: 1226±39 and 136±8, respectively. Error bars are standard errors (in most cases obscured by the data symbol).
Image published in: Cahu J et al. (2008)
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