Phosphoregulation of the cytokinetic protein Fic1 contributes to fission yeast growth polarity establishment

Abstract

Cellular polarization underlies many facets of cell behavior, including cell growth. The rod-shaped fission yeast Schizosaccharomyces pombe is a well-established, genetically tractable system for studying growth polarity regulation. S. pombe cells elongate at their two cell tips in a cell cycle-controlled manner, transitioning from monopolar to bipolar growth in interphase when new ends established by the most recent cell division begin to extend. We previously identified cytokinesis as a critical regulator of new end growth and demonstrated that Fic1, a cytokinetic factor, is required for normal polarized growth at new ends. Here, we report that Fic1 is phosphorylated on two C-terminal residues, which are each targeted by multiple protein kinases. Endogenously-expressed Fic1 phosphomutants cannot support proper bipolar growth, and the resultant defects facilitate the switch into an invasive pseudohyphal state. Thus, phosphoregulation of Fic1 links the completion of cytokinesis to the re-establishment of polarized growth in the next cell cycle. These findings broaden the scope of signaling events that contribute to regulating S. pombe growth polarity, underscoring that cytokinetic factors constitute relevant targets of kinases affecting new end growth.

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast

“…Pck1 is one of two protein kinase C enzymes in S. pombe ; it acts downstream of Rho1 GTPase to regulate septum deposition and participates in the cell wall integrity pathway ( Toda et al, 1993 ; Kobori et al, 1994 ; Arellano et al, 1999 ; Sánchez-Mir et al, 2014 ). Cdc15 phosphorylation was also modestly reduced by inhibiting the analog 3BrB-PP1 sensitive pck1 as2 (M744G) mutant ( Bohnert et al, 2020 ; Figure 1H ). We next combined pck1 as2 with the other three analog-sensitive mutations.…”

Stress-dependent inhibition of polarized cell growth through unbalancing the GEF/GAP regulation of Cdc42

“…In that case, media containing the drug was added and the chamber was left for 60 min in a 30 C incubator. We realized that the use of other ATP analogs such as the 1NM-PP1 or 3Br-PP1 had an inhibitory effect on the wild-type version of the Sty1 kinase, and probably other kinases (data not shown), as described elsewhere (Bohnert et al, 2020). For that reason, we strongly recommend the use of 3MB-PP1 with the analog sensitive version of Sty1 kinase, Sty1.T97A.…”

The fission yeast cytokinetic ring component Fic1 promotes septum formation

“…Because the Fic1-2A mutant eliminates phosphorylation on T178, we wondered whether disrupting the prolines required for Cyk3 binding around T178 might alter Fic1's phosphorylation status. We were especially cognizant of this possibility because T178 can be phosphorylated in vitro by CDK, a proline-directed kinase ( Bohnert et al, 2020 ). To examine whether these proline mutations affected Fic1 phosphorylation in vivo , we analyzed the gel mobilities of Fic1-P174,177A and Fic1-P176,179A.…”

“…We previously found that Fic1 is phosphorylated on two sites by multiple kinases ( Bohnert et al, 2020 ). Preventing phosphorylation at these sites produces defects in the establishment of normal cell polarity.…”