Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

Abstract

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–YFPC (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–YFPN 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.

A Dynamic Gene Regulatory Network Model That Recovers the Cyclic Behavior of Arabidopsis thaliana Cell Cycle

“…It seems that molecules such as WEE1 kinase and CDC25 phosphatase are not conserved. In A. thaliana , CDC25-like has phosphatase and arsenate-reductase functions [ 146 ], while A. thaliana WEE1 phosphorylates monomeric CDKA;1 in vitro [ 147 ], and Nicotiana tabacum WEE1 inhibits CDK activity in vitro [ 148 ]. However the lack of any obvious mutant phenotype of CDC25 or WEE1 loss-of-function mutants predicts that these genes are not involved in the regulation of a normal CC.…”

Spatial Regulation of Root Growth: Placing the Plant TOR Pathway in a Developmental Perspective

“…Recent data shows that the E2F-DP-RBR pathway is also active at the G2/M transition [ 20 ]. While CYC/CDK complexes are decisive actors of cell cycle progression, other kinases also play important roles including the DNA integrity checkpoint Wee1, the energy depletion sensor Sucrose non-fermenting-1-Related protein Kinase 1 (SnRK1), and the plant orthologue of the mammalian adenosine monophosphate-activated protein kinase (AMPK) [ 21 , 22 , 23 ]. Environmental cues and organ functional specificity will also add variations to the networking of the cell cycle interactome [ 24 , 25 ].…”

Drugs for Plant Chromosome and Chromatin Research

“…Its inhibition by aphidicolin [Spadari et al, 1982] has been used for cell-cycle synchrony and understanding DNA-replication checkpoints in plants [Francis, 2011;Cook et al, 2013].…”