The tobacco mitogen-activated protein kinase (MAPK) cascade, which includes MAPK NRK1/NTF6, positively regulates expansion of the cytokinetic machinery known as the phragmoplast, which is followed by the synthesis of cell plates for completion of cell division. However, molecular events lying between the MAPK and phragmoplast expansion were not known. Here, we show that NRK1/NTF6 phosphorylates the threonine residue at position 579 in NtMAP65-1a, a microtubule-associated (MT-associated) protein. Levels of phosphorylated NtMAP65-1 increase during late M phase of the cell cycle, when NRK1/NTF6 is activated. Phosphorylated NtMAP65-1 is concentrated at the equator of phragmoplast, as is NRK1/NTF6. Overexpression of mutant forms of NtMAP65-1a that cannot be phosphorylated by NRK1 delays progression of the M phase and phragmoplast expansion, also rendering phragmoplast structures resistant to an MT-depolymerizing drug. Phosphorylation of NtMAP65-1 by NRK1/NTF6 down-regulates its MT-bundling activity in vitro. These results suggest that phosphorylation of NtMAP65-1 by NRK1/NTF6 also reduces its MT-bundling activity in vivo, which enhances destabilization and turnover of MTs at the phragmoplast equator, perhaps facilitating phragmoplast expansion.[Keywords: MAP65/PRC1; MAPK; cytokinesis; microtubule; microtubule-associated proteins; phragmoplast] Supplemental material is available at http://www.genesdev.org. Cell division in eukaryotes requires dynamic changes in cytoskeletal structures that consist of microtubules (MTs) and microfilaments Jürgens 2005). During cytokinesis, the final critical step in the cell division, cells form a cytokinesis-specific apparatus (known as the central spindle in animals and the phragmoplast in plants) that plays a key role in cytokinesis (Field et al. 1999;. These structures develop from late anaphase to telophase between the two daughter nuclei, and consist of two bundles of antiparallel MTs. As cytokinesis proceeds, the central spindle becomes compacted in animal cells and the phragmoplast expands centrifugally in plant cells, and new membranes and/or cell walls are generated inside or outside the midzone of the central spindle or the phragmoplast to separate the two daughter cells from each other (Otegui et al. 2005). These dynamic processes appear to be mediated by the turnover of MTs, which involves the depolymerization of MTs and the polymerization of tubulin at the plus ends (Shelden and Wadsworth 1990;Asada et al. 1991;Hush et al. 1994;Straight and Field 2000). Recently, Austin et al. (2005) have reported that somatic-type phragmoplast MTs do not interdigitate at the cell plate mid-line and that distinct MT plus-end geometries are seen during the different stages of cytokinesis.The members of the MAP65/Ase1/PRC1 family (designated the MAP65 family herein)-including plant MAP65, yeast Ase1, and human PRC1-participate in the bundling of MTs (Chang-Jie and Sonobe 1993;Chan et al. 1999;Mollinari et al. 2002;Schuyler et al. 2003;

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