Cell cycle-dependent phosphorylation of IQGAP is involved in assembly and stability of the contractile ring in fission yeast

https://doi.org/10.1016/j.bbrc.2020.10.043Get rights and content

Highlights

  • CDK-dependent phosphorylation of IQGAP is involved in cytokinesis in S. pombe.

  • F-actin in the contractile ring is unstable in a phosphomimetic IQGAP rng2 mutant.

  • Phosphorylated Rng2 collaborates with myosin-II in assembly of the contractile ring.

Abstract

Cytokinesis is the final step in cell division and is driven by the constriction of the medial actomyosin-based contractile ring (CR) in many eukaryotic cells. In the fission yeast Schizosaccharomyces pombe, the IQGAP-like protein Rng2 is required for assembly and constriction of the CR, and specifically interacts with actin filaments (F-actin) in the CR after anaphase. However, the mechanism that timely activates Rng2 has not yet been elucidated. We herein tested the hypothesis that the cytokinetic function of Rng2 is regulated by phosphorylation by examining phenotypes of a series of non-phosphorylatable and phosphomimetic rng2 mutant strains. In phosphomimetic mutant cells, F-actin in the CR was unstable. Genetic analyses indicated that phosphorylated Rng2 was involved in CR assembly in cooperation with myosin-II, whereas the phosphomimetic mutation attenuated the localization of Rng2 to CR F-actin. The present results suggest that Rng2 is phosphorylated during CR assembly and then dephosphorylated, which enhances the interaction between Rng2 and CR F-actin to stabilize the ring, thereby ensuring secure cytokinesis.

Introduction

Cytokinesis bisects a cell into two daughters at the end of cell division following the completion of mitosis. Failed cytokinesis results in the unequal inheritance of cytoplasm, organelles, and chromosomes, which may cause cell death or heteroploidy [1]. In many eukaryotic cells, actin filaments (F-actin), actin-modulating proteins, and type II myosin (myosin-II) motors accumulate beneath the plasma membrane in the cell middle during cytokinesis. F-actin and myosin-II together assemble into a ring-like structure at the equatorial region, called the contractile ring (CR), and generate the force needed to cleave the cell by constricting the ring [2,3]. The precise control of the localization and activation of CR components is essential for CR assembly at the correct division site and for its constriction at the right timing.

The fission yeast Schizosaccharomyces pombe has provided an excellent model system for studying cytokinesis and CR formation over the decades [4]. In S. pombe, the CR is formed from dozens of small protein clusters called “cytokinetic nodes” [5]. In the late G2 phase, the fission yeast anillin-like protein Mid1 assembles as nodes around the medial cortex [6]. Mid1 then recruits core CR components to the nodes, including IQGAP Rng2, myosin-II heavy chain Myo2, myosin-II essential light chain Cdc4, the F-BAR protein Cdc15, and the formin Cdc12 [5].

In contrast to extensive studies on the localization of CR precursors in S. pombe and the underlying mechanisms, limited information is currently available on the mechanisms that activate CR components specifically at mitosis [7]. One of the few studies on this process was on the cell cycle-dependent phosphoregulation of Cdc15 activity; Cdc15 was shown to be inactivated during interphase by hyperphosphorylation and activated by hypophosphorylation in mitosis [8,9]. Active Cdc15 associates with Cdc12 and recruits Cdc12 to the CR [10]. In contrast to Cdc15, Cdc12 is activated during mitosis by its hyperphosphorylation [11]. The NDR kinase Sid2, a component of the septation initiation network (SIN), and cyclin-dependent kinase (CDK) Cdc2 contributes to the phosphorylation of Cdc12 [11,12]. Myo2 is also phosphorylated in the C-terminal region and dephosphorylated at early mitosis. Although this phosphoregulation of Myo2 is not required for CR assembly and cytokinesis, the phosphorylation of Myo2 promotes CR constriction [13].

Rng2, one of the core CR components, is the IQGAP-like protein in S. pombe and essential for CR assembly, and, thus, viability [14]. Similar to other IQGAPs, Rng2 is a large (1489 a.a, 172-kDa) scaffolding protein that contains multiple functional domains: an N-terminal calponin homology domain (CHD), seven IQ motifs, a rasGAP-related domain (GRD), and a C-terminal Ras-GAP domain (Fig. 1A). The CHD of Rng2 associates with all F-actin structures in S. pombe cells (actin patches, cables, and the CR). Rng2 physically interacts with Mid1, is required for the subsequent recruitment of myosin-II Myo2 to cytokinetic nodes [15,16], and maintains the F-actin-independent localization of myosin-II in the CR [17]. Rng2 localizes to the division site mainly by its C-terminal region, which interacts with the N-terminal region of Mid1 [15], whereas the CHD is not essential for the recruitment of myosin-II by Rng2 [18]. A short N-terminal moiety of Rng2 containing the CHD, Rng2Ns (1–300 a.a.) only localizes to the CR in an F-actin-dependent manner [19], implying a specific interaction with CR F-actin.

A comprehensive phosphoproteomic analysis revealed that IQGAP Rng2 is phosphorylated during mitosis [20,21]. However, the significance of the phosphorylation of Rng2 remains unclear. In the polymorphic fungus Candida albicans, the IQGAP Iqg1 is also a component of the CR and phosphorylated by CDK [22]. The replacement of wild-type Iqg1 with a non-phosphorylatable mutant caused both the premature assembly and delayed disassembly of the CR, and weakened its interaction with the formins Bni1 and Bnr1, resulting in defective cytokinesis [22]. In the budding yeast Saccharomyces cerevisiae, the phosphorylation of IQGAP Iqg1 is regulated by CDK and Cdc14 phosphatase [23,24]. Similar to C. albicans Iqg1, preventing the phosphorylation of S. cerevisiae Iqg1 facilitates CR assembly and stabilizes the ring. Thus, these findings on IQGAP proteins in other yeasts prompted us to hypothesize that the activity of Rng2 is also regulated by CDK-dependent phosphorylation.

In the present study, we examined the role of the phosphorylation of Rng2 in CR assembly and cytokinesis by examining the phenotypes of the non-phosphorylatable and phosphomimetic mutants of Rng2. The CR was fragile against actin depolymerization in cells expressing only the phosphomimetic mutant Rng2. The phosphomimetic mutation of Rng2 reduced the F-actin-dependent localization of Rng2 to the CR. Moreover, the non-phosphorylatable rng2 mutant genetically interacted with the thermosensitive cytokinetic mutant myo2-E1, and the corresponding double mutant showed defects in CR assembly and cytokinesis. These results suggest that the phosphorylation of Rng2 from metaphase to early anaphase is involved in the assembly of the CR in collaboration with Myo2, whereas the dephosphorylation of Rng2 after anaphase enhances the interaction of Rng2 with CR F-actin to stabilize the ring, thereby ensuring secure cytokinesis.

Section snippets

Yeast strains and cell culture

The yeast strains and plasmids used in the present study are listed in Tables S1 and S2, respectively. Cells were grown in rich medium containing yeast extract (YE) or in minimal medium (EMM). We used the leu1-32 strain (h- leu1-32) as wild-type S. pombe. To observe the CR, we used a strain expressing the myosin regulatory light chain tagged with the red fluorescent protein mCherry (rlc1-mCherry ade- ura4-D18 leu1-32). Pseudo-phosphorylation mutations were introduced on plasmids by

The phosphorylation of Rng2 contributes to maintaining the CR

S. pombe CDK Cdc2 preferably phosphorylates the minimal (S/T-P) or perfect CDK consensus site (S/T-P-X-R/K, in which X is any amino acid) [[26], [27], [28]]. In Rng2, there were 12 CDK-consensus phosphorylation sites (Fig. 1A), most of which show high phosphorylation potentials according to the NetPhos scores (Fig. 1B) [29]. Six of the sites were concentrated in the neck region after CHD (Fig. 1C). This neck region was predicted to be a highly disordered structure [30] (Fig. S1), and, hence, we

Discussion

In the present study, the phosphomimetic mutation induces the fragility of the CR against actin depolymerization. On the other hand, the transient phosphorylation of Rng2 appears to be important for CR assembly in collaboration with myosin-II Myo2. Therefore, Rng2 might be phosphorylated from metaphase by CDK Cdc2 and becomes active in assembling the CR, whereas its actin-binding activity is weakened and then dephosphorylated after anaphase to bind to and stabilize CR F-actin.

In the budding

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers 19037004 and 20770150 to KN and 24770177 to MT.

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