244 M.Sasabe·Y.Machida
against Thr-579-phosphorylated NtMAP65-1 have revealed that NtMAP65-1
is phosphorylated at this site in vivo. Because PRC1 (a mammalian MAP65
homolog) is phosphorylated by Cdc2 (Jiang et al. 1998), we also examined
whether NtMAP65-1a can be also phosphorylated by CDKs from BY-2 cells
in vitro. We found that the sites phosphorylated by CDKs were different from
that phosphorylated by NRK1 (Fig. 5A; Sasabe et al. 2006).
Characterization of NtMAP65-1 phosphorylated by MAPK.Insynchro-
nized BY-2 cells, NtMAP65-1 phosphorylated at Thr-579 accumulates at
late M phase, although the total amount of NtMAP65-1 does not change.
Such a pattern of phosphorylation is consistent with the pattern of NACK1
accumulation and NPK1, NQK1/NtMEK1, and NRK1/NTF6 activation. We
have found that a GFP-NtMAP65-1a fusion protein localizes on all kinds
of MT structures, including cortical MTs, the preprophase band, spindles,
and the phragmoplast (our unpublished observation). Immunostaining with
NtMAP65-1 antibodies also revealed NtMAP65-1 on various MT structures
throughout the cell cycle. Interestingly, NtMAP65-1 phosphorylated on Thr-
579 is concentrated at the equator of the phragmoplast along with other
components of the NACK-PQR pathway, although NtMAP65-1 can be found
throughout the entire phragmoplast (Fig. 5B; Sasabe et al. 2006). These find-
ings suggest that Thr-579 of NtMAP65-1 is phosphorylated by NRK1/NTF6 at
the phragmoplast midzone during cytokinesis.
Fig. 5 NtMAP65-1 acts downstream of the NACK-PQR pathway.AA schematic repre-
sentation of NtMAP65-1a. The coiled-coil motif (CC) and the motif that is conserved in
the MAP65 family (CM) are described. Sites of phosphorylation (P) by NRK1 and CDKs
are also indicated.BSubcellular localization of NtMAP65-1 (top) and NtMAP65-1 phos-
phorylated on Thr-579 (bottom) at telophase in BY-2 cells. BY-2 cells were triple-stained
with mouse antibodies against a-tubulin (green), rabbit antibodies against NtMAP65-1 or
NtMAP65-1 phosphorylated on Thr-579 (red), and DAPI (blue).CDark-field micrographs
of MTs bundled in vitro by NtMAP65-1 (left) and NRK1-phosphorylated NtMAP65-1
(right). The MT-bundling activity of NtMAP65-1 was decreased by phosphorylation with
NRK1