Biomedical subjects
Ryuichi Nishihama
Publications and source records attributed to Ryuichi Nishihama.
NQK1/NtMEK1 is a MAPKK that acts in the NPK1 MAPKKK-mediated MAPK cascade and is required for plant cytokinesis.
The tobacco protein kinase NPK1 is a MAPKKK that regulates formation of the cell plate during cytokinesis. In the present study, we have identified tobacco NQK1/NtMEK1 and NRK1 as a MAPKK and a MAPK, respectively, downstream of NPK1. NQK1/NtMEK1 complements the mutation in the PBS2 MAPKK gene of yeast in a manner that depends on both NPK1 and its activator, NACK1, a kinesin-like protein. Active NPK1 and NQK1/NtMEK1 phosphorylate and activate NQK1/NtMEK1 and NRK1, respectively. Both NQK1/NtMEK1 and NRK1, as well as NPK1, are activated at the late M phase of the cell cycle in tobacco cells, and they are rapidly inactivated by depolymerization of phragmoplast microtubules. These results suggest the existence of a MAPK cascade that consists of NPK1, NQK1/NtMEK1, and NRK1 and functions in a process related to the architecture of phragmoplasts at the late M phase of the cell cycle. Overexpression of kinase-negative NQK1/NtMEK1 in tobacco cells generates multinucleate cells with incomplete cross-walls. Arabidopsis plants with a mutation in the ANQ1 gene, an ortholog of NQK1/NtMEK1, display a dwarf phenotype, with unusually large cells that contain multiple nuclei and cell-wall stubs in various organs. In addition, anq1 homozygotes set fewer flowers and produce large and malformed pollen grains with a tetrad structure. Thus, NQK1/NtMEK1 (ANQ1) MAPKK appears to be a positive regulator of plant cytokinesis during meiosis as well as mitosis.
Control of plant cytokinesis by an NPK1-mediated mitogen-activated protein kinase cascade.
Cytokinesis is the last essential step in the distribution of genetic information to daughter cells and partition of the cytoplasm. In plant cells, various proteins have been found in the phragmoplast, which corresponds to the cytokinetic apparatus, and in the cell plate, which corresponds to a new cross wall, but our understanding of the functions of these proteins in cytokinesis remains incomplete. Reverse genetic analysis of NPK1 MAPKKK (nucleus- and phragmoplast-localized protein kinase 1 mitogen-activated protein kinase kinase kinase) and investigations of factors that might be functionally related to NPK1 have helped to clarify new aspects of the mechanisms of cytokinesis in plant cells. In this review, we summarize the evidence for the involvement of NPK1 in cytokinesis. We also describe the characteristics of a kinesin-like protein and the homologue of a mitogen-activated protein kinase that we identified recently, and we discuss possible relationships among these proteins in cytokinesis.
Expansion of the cell plate in plant cytokinesis requires a kinesin-like protein/MAPKKK complex.
The tobacco mitogen-activated protein kinase kinase kinase NPK1 regulates lateral expansion of the cell plate at cytokinesis. Here, we show that the kinesin-like proteins NACK1 and NACK2 act as activators of NPK1. Biochemical analysis suggests that direct binding of NACK1 to NPK1 stimulates kinase activity. NACK1 is accumulated specifically in M phase and colocalized with NPK1 at the phragmoplast equator. Overexpression of a truncated NACK1 protein that lacks the motor domain disrupts NPK1 concentration at the phragmoplast equator and cell plate formation. Incomplete cytokinesis is also observed when expression of NACK1 and NACK2 is repressed by virus-induced gene silencing and in embryonic cells from Arabidopsis mutants in which a NACK1 ortholog is disrupted. Thus, we conclude that expansion of the cell plate requires NACK1/2 to regulate the activity and localization of NPK1.
The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein.
The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.