Dueling Kinases Regulate Cell Size at Division through the SAD Kinase Cdr2

Abstract

Summary Cell size control requires mechanisms that integrate cell growth and division. Key to this integration in fission yeast is the SAD family kinase Cdr2, which organizes a set of cortical nodes in the cell middle to promote mitotic entry through Wee1 and Cdk1 [1-3]. Cdr2 is inhibited by a spatial gradient of the DYRK kinase Pom1 emanating from cell tips in a cell size-dependent manner [2, 3], but how the Pom1 gradient inhibits Cdr2 activity during cell growth is unknown. Here, we show that Pom1 acts to prevent activation of Cdr2 kinase activity by the CaMKK Ssp1. We found that Ssp1 activates Cdr2 through phosphorylation of a conserved threonine residue (Thr166) in the activation loop of the Cdr2 N-terminal kinase domain both in vitro and in cells. The levels of this activating phosphorylation increased with cell cycle progression, and genetic epistasis demonstrated that Ssp1 promotes mitotic entry through Cdr2. Intriguingly, Pom1 phosophorylated the C-terminal domain (CTD) of Cdr2, and this modification reduced Cdr2-T166 phosphorylation by Ssp1. These findings show how activation of the conserved mitotic inducer Cdr2 is integrated with an inhibitory spatial gradient to ensure proper cell size control at mitosis.

Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1*

“…This is important because Cdr2 is mislocalized in pom1⌬ cells (2,3), raising the possibility that Pom1 regulates mitotic entry through localization of Cdr2. However, data from our study, in combination with previous work from our group and others (8,9), suggest that Pom1 phosphorylates the Cdr2-CTD to inhibit Cdr2 kinase activity and prevent mitotic entry. This regulation of Cdr2 enzyme activity is separate from Pom1 regulation of Cdr2 localization, which appears to involve additional phosphorylation sites in membrane-binding region of Cdr2 (10).…”

“…In contrast to this complex cell polarity network, Pom1 has been proposed to regulate mitotic entry through a single protein, the SAD kinase Cdr2, which is directly phosphorylated by Pom1 in vitro (2,8,9). Indeed, we identified a cluster of Pom1-dependent phosphorylation sites in the C-terminal domain of Cdr2 (Table I).…”

“…Cdr2 has been shown to be a direct substrate of Pom1 in vitro (2,8,9), while the other top targets represent novel substrates for For each phosphopeptide, the log2-transformed heavy/light ratio is given for pom1-as and wild type strains as indicated. Abbreviations: # ϭ phosphorylation; * ϭ oxidation.…”

“…Protein Purification and In Vitro Kinase Assays-Recombinant Pom1 and kinase-dead Pom1(K728R) mutants were purified from Escherichia coli as previously described (9). The same methods were used to purify recombinant Tea4(1-296) and Mod5(28 -495) fragments.…”

“…Second, Pom1 phosphorylates two regions of the protein kinase Cdr2. Phosphorylation of Cdr2 C terminus is proposed to prevent mitotic entry by inhibiting Cdr2 kinase activity (8,9), while phosphorylation near membrane-binding motifs of Cdr2 promotes medial cell division by inhibiting localization of Cdr2 at cell tips (10). It has been unclear if Cdr2 represents the only cell cycle target of Pom1 kinase activity, and no cell polarity targets of Pom1 have been identified.…”