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A signaling module of NIMA-related kinases (Neks) regulates two kinesins, Mklp2 and Kif14, to spatiotemporally coordinate their subcellular localizations and activities. This is important for faithful completion of cytokinesis and reveals novel mechanisms by which Neks regulate late mitosis.

Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k max = 0.033 ± 0.006 sec −1 and K I = 19.7 ± 7.2 μM. Structure determination of acivicin-modified HpGT (1.7 Å; R factor =17.9%; R free =20.8%) demonstrates that acivicin is accommodated within the γ-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole. Keywordsγ-glutamyltranspeptidase; Ntn-hydrolase; glutathione; acivicin; x-ray crystallography H. pylori γ-glutamyltranspeptidase (HpGT) is a γ-glutamyl hydrolase with broad substrate specificity (1,2), and is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily (3,4). The inactive precursor undergoes an intramolecular autoprocessing event, generating the mature and catalytically active heterotetramer. A conserved threonine residue, Thr 380, serves as the N-terminal nucleophile and is required for both maturation and enzymatic activity (1).HpGT has been shown to degrade extracellular glutathione and glutamine, providing a growth advantage to the bacterium within its microenvironment (2,5,6). Similarly, upregulation of human γ-glutamyltranspeptidase in cancer is thought to help supplement these rapidly dividing cells with essential amino acid precursors for glutathione and protein biosynthesis (7,8). In mammalian systems, γ-glutamyltranspeptidase has been shown to be critical for the transport of cysteine for use in protein and glutathione biosynthesis (9,10). The enzyme is required for normal glutathione metabolism, initiating extracellular glutathione degradation. Subsequent steps lead to the cellular uptake of the composite amino acids of glutathione: glutamate, cysteine, and glycine (11,12).Acivicin is a com...

a These authors contributed equally to this work. b Current address: Abcam, AbstractChromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein, Sid4, at the SPB, and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Polo-like kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1's SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast, Schizosaccharomyces pombe. Because cell cycle regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog, Cdc15, controls Cdc15 oligomerization state, localization, and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR with the bulk of phosphorylation being constitutive. In vitro, casein kinase 1 (CK1) Hhp1 and Hhp2 can phosphorylate 17 sites and Cdk1 the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 CR accumulation. Mutations that eliminated CK1 phosphorylation sites allowed CR sliding, and phosphomimetic substitutions at these sites reduced Imp2 protein levels and slowed CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive with milder effects on localization and function.

During cell division, the timing of mitosis and cytokinesis must be ordered to ensure that each daughter cell receives a complete, undamaged copy of the genome. In fission yeast, the septation initiation network (SIN) is responsible for this coordination, and a mitotic checkpoint dependent on the E3 ubiquitin ligase Dma1 and the protein kinase CK1 controls SIN signaling to delay cytokinesis when there are errors in mitosis. The participation of kinases and ubiquitin ligases in cell cycle checkpoints that maintain genome integrity is conserved from yeast to human, making fission yeast an excellent model system in which to study checkpoint mechanisms. In this review, we highlight recent advances and remaining questions related to checkpoint regulation, which requires the synchronized modulation of protein ubiquitination, phosphorylation, and subcellular localization.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N‐terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation‐induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high‐affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. CK1 kinases have highly similar catalytic domains, plus a conserved extension to that kinase domain that is important for enzyme stability and activity. In contrast to the catalytic domains, the C‐terminal tails of CK1 family members diverge in sequence and length; however, they all appear to serve as substrates of autophosphorylation. The autophosphorylated tails are proposed to inhibit kinase activity by acting as pseudosubstrates. In addition to C‐terminal autophosphorylation, autophosphorylation of the CK1□ kinase domain has been detected, but its effect on CK1 activity and cellular function has never been explored. Here, we addressed the role of kinase domain autophosphorylation in human CK1□ and CK1□, as well as Hhp1 and Hhp2, their homologues in Schizosaccharomyces pombe. In each case, we found that autophosphorylation of a conserved threonine residue in the kinase domain inhibited enzyme activity. This site resides in the mobile L‐EF loop proximal to the active site, distinct from the well‐characterized T‐loop autophosphorylation that occurs in other kinase families. We found that yeast and human enzymes with phosphoablating mutations at this site are hyperactive in vitro. In vivo, mutation of this site protects yeast cells from heat shock, indicating a change in substrate profile, a prediction we are currently testing using quantitative phosphoproteomics. We have also found that autophosphorylation of the kinase domain may affect autophosphorylation of the C‐terminus, so there is likely interplay between the two different modalities of CK1 autoregulation that ultimately determines the extent to which substrates are phosphorylated. We propose that phosphorylation on the L‐EF loop prevents substrate docking with the kinase domain by shielding the positively charged substrate binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L‐EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo. Support or Funding Information This work was supported by R35‐GM131799 (to KLG) and T32‐CA119925 (to SNC).