Cell size-dependent regulation of Wee1 localization by Cdr2 cortical nodes

Abstract

Cell size control requires mechanisms that link cell growth with Cdk1 activity. In fission yeast, the protein kinase Cdr2 forms cortical nodes that include the Cdk1 inhibitor Wee1, along with the Wee1-inhibitory kinase Cdr1. We investigated how nodes inhibit Wee1 during cell growth. Biochemical fractionation revealed that Cdr2 nodes were megadalton structures enriched for activated Cdr2, which increases in level during interphase growth. In live-cell TIRF movies, Cdr2 and Cdr1 remained constant at nodes over time, but Wee1 localized to nodes in short bursts. Recruitment of Wee1 to nodes required Cdr2 kinase activity and the noncatalytic N-terminus of Wee1. Bursts of Wee1 localization to nodes increased 20-fold as cells doubled in size throughout G2. Size-dependent signaling was due in part to the Cdr2 inhibitor Pom1, which suppressed Wee1 node bursts in small cells. Thus, increasing Cdr2 activity during cell growth promotes Wee1 localization to nodes, where inhibitory phosphorylation of Wee1 by Cdr1 and Cdr2 kinases promotes mitotic entry.SummaryCells turn off the mitotic inhibitor Wee1 to enter into mitosis. This study shows how cell growth progressively inhibits fission yeast Wee1 through dynamic bursts of localization to cortical node structures that contain Wee1 inhibitory kinases.

A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1

“…Consistent with these biochemical results, only over-expression of Cdr1 but not of Cdr2 resulted in reduced cell size in cdr1∆cdr2∆ cells ( Fig 1C), consistent with previous results in wild type cells (Russell and Nurse, 1987;Breeding et al, 1998). Phosphorylation of Wee1 in fission yeast cells was reduced in the catalytically inactive mutant cdr1(K41A) (Fig 1D), similar to cdr1∆ cells (Allard et al, 2018). Given the role of Cdr1 in regulating Wee1 kinase activity, along with open questions regarding its underlying mechanism, we investigated this pathway further.…”

“…This localization requires Wee1's non-catalytic N-terminus, which can be phosphorylated by Cdr2 (Kanoh and Russell, 1998;Allard et al, 2018). In kinasedead cdr2 mutants, Wee1 localizes to nodes in extremely short bursts (< 2 seconds) (Allard et al, 2018). As the first step towards inhibition, we propose that Cdr2 phosphorylates the Wee1 Nterminus to slow the off-rate for Wee1 release, thereby "trapping" Wee1 in the node.…”

“…Cdr1 and Wee1 both localize to cortical nodes in the cell middle (Martin and Berthelot-Grosjean, 2009;Moseley et al, 2009;Allard et al, 2018). These nodes are formed by Cdr2 oligomers that are required for Cdr1 and Wee1 recruitment (Martin and Berthelot-Grosjean, 2009;Moseley et al, 2009;Allard et al, 2018).…”

“…Our results combined with past work suggest a two-step mechanism for regulation of a Wee1 molecule at nodes ( Fig 5E). Wee1 localizes to nodes in transient bursts that last between 5-15 seconds (Allard et al, 2018). This localization requires Wee1's non-catalytic N-terminus, which can be phosphorylated by Cdr2 (Kanoh and Russell, 1998;Allard et al, 2018).…”

“…Wee1 localizes to nodes in transient bursts that last between 5-15 seconds (Allard et al, 2018). This localization requires Wee1's non-catalytic N-terminus, which can be phosphorylated by Cdr2 (Kanoh and Russell, 1998;Allard et al, 2018). In kinasedead cdr2 mutants, Wee1 localizes to nodes in extremely short bursts (< 2 seconds) (Allard et al, 2018).…”