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Figure 2


Fig. 2. Several protein kinases phosphorylate AtMAP65-1. (A) Effect of protein kinase inhibitors on AtMAP65-1 phosphorylation in vitro. Recombinant AtMAP65-1 was phosphorylated using M-phase extract (sampling point 4 as in Fig. 1A) without inhibitor (Control) or in the presence of 20 mM DMAP, 5 µM K252a, 5 µM staurosporine and 100 µM olomoucine. Each lane contained an equal amount of protein as shown by the amido-black staining. (B) AtMAP65-1 is phosphorylated by microtubule associating kinases. Colloidal silver staining and autoradiogram of phosphorylation assays using M-phase extract (lane 1) and a microtubule-associated protein preparation (lane 2). (C) AtMAP65-1 is phosphorylated by CDKs and MAPKs. Cyclin-dependent kinases were pulled down using pSuc1 bound beads, anti-cdc2a and anti-cdc2b. MAP kinases were immunoprecipitated with anti-MPK4 and anti-MPK6. Kinase activity was checked using histone 1 as a substrate for cyclin-dependent kinases and using myelin basic protein (MBP) as the substrate for MAP kinases. (D) Localisation of GFP:AtMAP65-1 in control cells. (Interphase (1), metaphase (2), anaphase (3) telophase (4). (E) Localisation of GFP:AtMAP65-1 in cells treated for 10 minutes with the general protein kinase inhibitor DMAP. Interphase (1), metaphase (2), anaphase (3) and telophase (4). (F) Localisation of GFP:AtMAP65-1 in cells treated for 10 minutes with the CDK inhibitor olomoucine. Prometaphase (1), metaphase (2 and 3). (G) Three consecutive movie frames (taken 2 minutes apart) of a cell treated with the phosphatase inhibitor okadaic acid. The images were recorded after 15 minutes of treatment. Note the GFP:AtMAP65-1 signal disappears and the phragmoplast does not expand in this time. (H) Localisation of AtMAP65-1 (green signal) and DNA (blue signal) in the phragmoplast of a BY-2 cell line harbouring a non-degradable form of cyclin B1 under the control of the dexamethasone-inducible promoter before induction (1) and after 16 hours induction with dexamethasone (2).





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