Factors Upstream of NPK1 MAPKKK

Activation of the MAP kinase pathway consisting of NPK1, NQK1 and NRK1 is observed only during the late M-phase of the cell division cycle, although the amounts of these protein kinases do not change during the M-phase. Two proteins, designated NACK1 and NACK2, have been identified as proteins activating NPK1 in yeast and have been shown to interact with NPK1 in vitro and vivo (Nishihama et al. 2002). Both proteins bind NPK1 to activate it. They are members of the kinesin-like protein family, which are motor proteins directed towards the plus end of microtubules. Overexpression of the truncated NACK1 protein, which lacks the motor domain, resulted in multinucleation and failure to complete cell plate development. In cells where this occurs, the localization of NPK1 to the phragmoplast equator is also disrupted, suggesting that NACK1 is required for the proper localization of NPK1 and that the activation of NPK1 in a limited region is important for phragmoplast-mediated cytokinesis.

The levels of mRNAs of NACK1 and NACK2 increase at the beginning of M-phase and decrease on exit from cytokinesis (Nishihama et al. 2002). The accumulation profile of NACK1 protein is similar to that of its transcripts. This cell cycle-dependent expression of NACK1 and NACK2 seems to be crucial in the regulation of the NPK1 activity that is required for phrag-moplast expansion. Transcription of both NACK1 and NACK2 is regulated by a cis-element, designated the MSA element on its promoter, which mediates activation by a specific class of MYB proteins (Ito et al. 2001). These MYB proteins have three imperfect repeats in the DNA binding domain, as in animal c-MYB proteins, whereas the vast majority of plant MYB proteins have two repeats. At least one member of the MYB protein family, called NtMYBA2, is activated through phosphorylated by cyclin-dependent protein kinase (CDK; Araki et al. 2004). Since NPK1 and NACK1 (and NACK2) have potential sites of phosphorylation by CDKs (Nishihama et al. 1997, 2002), activation of NPK1 by protein-protein interaction with NACK1 might be controlled through phosphorylation by CDKs during anaphase.

Arabidopsis homologs of NACK1 and NACK2 have been identified by conventional molecular cloning and have been designated AtNACK1 and At-NACK2, respectively (Nishihama et al. 2002). These genes have also been identified by formal genetic studies and named HINKEL and TETRASPORE/ STUD, respectively (Hulskamp et al. 1997; Spielman et al. 1997; Strompen et al. 2002; Yang et al. 2003). The mutation in AtNACK1/HINKEL, transcripts of which can be detected in a number of somatic cells of Arabidopsis plants, causes defects in cell division with multinucleation and the forma tion of cell wall stubs at various developmental stages from embryogenesis to postembryonic development. The mutation in AtNACK2/TETRASPORE/ STUD, transcripts of which are detected mainly in male gametes (Tanaka et al. 2004), however, causes only the generation of abnormal pollen. Double mutations in AtNACK1 and AtNACK2 cause both male and female gameto-phytic lethality, suggesting partial redundancy of AtNACK1 and AtNACK2 in cytokinesis during gamete formation (Tanaka et al. 2004). In contrast to the genomes of tobacco and Arabidopsis, the rice genome contains only one gene homologous to NACK1, designated DBS1 (Sazuka et al. 2005). The dbs1 mutant also shows defective cytokinesis, suggesting the conserved function of NACK-related genes among higher plants. The mutation observed in dbs1 is a single-nucleotide substitution at the splicing donor site, decreasing the amount of mature spliced mRNA for DBS1, which produces a leaky pheno-type.

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