Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa

Reversible phosphorylation of serine/threonine residues of cell cycle-regul atory proteins is one of the key molecular mechanisms controlling eukaryoti c cell division. In plants, the protein kinase partners (i.e. p34(cdc2/CDC2 8)-related kinases) have been extensively studied, while the role of counte r-acting protein phosphatases is less well understood. We used endothall (E T) as a cell-permeable inhibitor of serine/threonine-specific protein phosp hatases to alter cytological and biochemical characteristics of cell divisi on in cultured alfalfa cells. A high concentration of ET (10 and 50 mu m) i nhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low conc entration (1 mu m) of ET-treatment primarily reduced the PP2A activity. Hig h concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In con trast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exp osure of cells to 1 mu m ET resulted in disturbance of preprophase band for mation, increase in the number of nuclei with prophase microtubule assembly , premature polarisation of the spindle, and abnormal phragmoplast maturati on. Under the same conditions, the ET-treated cells exhibited an early incr ease in cdc2MsF kinase activity. These results suggest that PP2A contribute s to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on bo th PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.