RESUMEN
In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 interacting partner 1 (SKIP1). Furthermore, the AtWEE1-green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1-YFP(C) (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1-YFP(N) negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome.
Asunto(s)
Ciclo Celular/fisiología , Proteínas de Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/citología , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/genética , Mitosis , Proteínas de Plantas/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Serina-Treonina Quinasas/genética , Nicotiana/citología , Nicotiana/enzimologíaRESUMEN
The NME1 gene represents the prototypical metastasis suppressor, whose expression inhibits cell motility and metastasis without impact on primary tumor growth in a number of different human cancers. This report outlines our recent efforts to define the molecular mechanisms through which NME1 both suppresses cell motility and promotes genomic integrity in the setting of human melanoma. Forced NME1 expression in a variety of melanoma-derived cell lines was shown to induce dynamic changes in cell morphology and reorganization of the actin cytoskeleton, with formation of a network of thick stress fibers and assembly of fibronectin fibrils at large focal adhesions. Moreover, NME1 expression results in adhesion reprogramming through an impact on integrin repertoire and focal adhesion dynamics. Having previously demonstrated that NME1 expression promotes repair of DNA damage induced by ultraviolet radiation (UVR) in both yeast and mammalian cells, probably via the nucleotide excision repair pathway, we have more recently demonstrated that NME1 is rapidly recruited to double-strand breaks. This preliminary result represents the first evidence of direct interactions between NME1 and DNA in the context of DNA repair and has set the stage for current efforts to probe its functional interactions with double-strand break repair pathways. Discussed herein are molecular models to explain the interactions of NME1 with such diverse cellular functions as cell motility and DNA repair, potentially through its nucleoside diphosphate kinase and 3'-5' exonuclease activities.