bioRxiv Science⌕ Search

bioRxiv · 10.1101/2022.04.01.485959

A dual mechanism of APC/C inhibition by MAP kinases

Abstract

Mitotic anaphase onset is a key cellular process that is tightly regulated by multiple kinases. The involvement of mitogen-activated protein kinases (MAPKs) in this process has been established long ago in Xenopus egg extracts. However, despite its importance, it is still unclear which MAPK(s) is actually involved, this impedes the further understanding of the regulatory cascade. In this study, we first demonstrated that the involvement of MAPKs in mitotic anaphase onset regulation is evolutionarily conserved in the fission yeast (Schizosaccharomyces pombe). Then, we found that two of the three fission yeast MAPK signaling pathways act in concert to restrain anaphase-promoting complex/cyclosome (APC/C) activity upon activation of the spindle assembly checkpoint (SAC). The first pathway involves the phosphorylation of Mad2, a component of the core mitotic check complex (MCC), by MAPK Sty1, which enhances the tight binding of MCC to APC/C. The second pathway involves MAPK Pmk1 phosphorylation of Slp1Cdc20, the fission yeast homologue of Cdc20 and the co-activator of APC/C, which promotes the degradation of Slp1Cdc20. Both phosphorylation events are required to sustain mitotic arrest in response to spindle defects. These results clarified a detailed regulation cascade of the ubiquitous MAPK signaling in spindle checkpoint activation, APC/C inhibition and anaphase entry, which is vital for accurate chromosome segregation and cell viability.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sun, L., Bai, S., Chen, J.-l., Deng, D.-j., Luo, Z.-q., Wang, Y., JIN, Q.. 2022-04-03. A dual mechanism of APC/C inhibition by MAP kinases. https://doi.org/10.1101/2022.04.01.485959

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

A dynamic phosphoproteome reveals stage-specific regulation of cytokinesis in Leishmania

Leishmania parasites undergo extensive morphological and metabolic remodelling during their transition from motile insect promastigotes to non-motile intracellular amastigotes, yet how dynamic protein kinase signalling drives this process remains poorly understood. Here, we present a high-resolution temporal phosphoproteome of L. mexicana promastigote to axenic amastigote differentiation, profiling 16,495 phosphosites on 3,101 proteins across seven developmental time points and identifying 5,795 dynamically regulated sites. This temporal map reveals stage-specific signalling programs, including regulated sites in MPK2 and non-regulated sites in MRK1, protein kinases known to be essential for amastigote differentiation. Using analogue-sensitive (AS) chemical genetics, we show that acute inhibition of either kinase in axenic amastigotes disrupts the spatial coordination of cleavage furrow ingression with nuclear and kinetoplast segregation, revealing essential cytokinesis roles obscured in constitutive gene deletions. Despite generating similar cytokinesis phenotypes, genetic over-expression and cellular localisation analyses demonstrate that MPK2 and MRK1 act through spatially segregated signalling pathways. Time-resolved phosphoproteomics upon MPK2 inhibition places its activity upstream of cytoskeletal remodelling networks. Importantly, we show that the stage-specific requirement for MPK2 is defined by the parasite's morphology: while motile promastigotes tolerate MPK2 inhibition, genetic ablation of flagellar motility sensitises them to loss of MPK2 activity. Thus, flagellar motility acts as a mechanical buffer during promastigote division, explaining why non-motile amastigotes are vulnerable to MPK2 inhibition. Together, our findings provide a systems level framework for Leishmania signalling and demonstrate that cellular architecture and biomechanics dictate protein kinase dependency across developmental stages of the parasite.

cell biology↗

Single-cell Transcriptome-Proteome-PTMome(x8)-Metabolome (scTPPM) co-profiling decodes multimodal regulation during mouse oocyte meiosis

Single-cell multi-omics technology is crucial for comprehensive characterization of cellular molecular features. Despite recent advances in single-cell multi-omics approaches, co-profiling of nucleic acids, proteins, metabolites, and post-translational modifications (PTMs) within the same cell individuals remains major challenges. Here, we developed a single-cell transcriptome, proteome, PTMome, and metabolome (scTPPM) profiling approach, enabling simultaneous analysis of transcriptomes, proteomes, eight types of PTMs (e.g., phosphorylation, methylation, ubiquitination, O_GlcNAc, acetylation, crotonylation, lactylation, and succinylation), and metabolomes in individual cells. We demonstrated the scTPPM's performance in tumor cell lines and oocyte samples, reaching total identification and quantification of 27,668 genes, 4,847 proteins, 1,508 PTM sites, and 680 metabolites in single mouse oocytes. We profiled previously-uncharacterized dynamic crosstalk among the entire metabolic pathways from metabolic enzyme expression and regulation to metabolite-mediated post-translational modifications during oocyte maturation. In summary, scTPPM provided an effective workflow for multi-omics co-profiling in single cells, enabling multi-layered insights into complex regulatory network.

cell biology↗

Sequence context and partner availability specify divergent cell fates from a single transcription factor

Transcription factors drive the gene regulatory networks that control developmental transitions, yet which of their many targets a cell uses remains unresolved. Expressing Cebp, Cebp{beta} or Cebp{delta} identically, alone or with Oct4, Sox2, Klf4 and cMyc, gave four outcomes: adipocyte from fibroblasts and neural stem cells, trophectoderm from embryonic stem cells, and myeloid from B cells. Raising Cebp levels changed how many sites it reached, not which sequence it read: clustered motifs maintained somatic enhancers, while spillover onto low-copy sites created de novo enhancers carrying composites of Cebp and cell-type-restricted partners. In fibroblasts, Cebp also redistributed Oct4, Sox2 and Klf4 from early targets, blocking iPSC formation. Supplying the predicted partners rewrote the output: Spi1 redirected Cebp into parallel adipogenic and myeloid programs, via a transient PpargCD45 state using separate enhancers for each; TFAP2C abolished adipogenic conversion. Motif density and partner composites therefore assemble a factor's instructive output in the receiving cell.

cell biology↗