Jungseog Kang

Phospho-Regulation of Kinetochore-Microtubule Attachments by the Aurora Kinase Ipl1p

The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known. To identify Ipl1p targets, we first purified 28 kinetochore proteins from yeast protein extracts. These studies identified five previously uncharacterized kinetochore proteins and defined two additional kinetochore subcomplexes. We then used mass spectrometry to identify 18 phosphorylation sites in 7 of these 28 proteins. Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing us to identify a consensus phosphorylation site for an Aurora kinase. Our systematic mutational analysis of the Ipl1p phosphorylation sites demonstrated that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo.

Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)–related protein Sli15 during chromosome segregation

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1–Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome–spindle interactions or in the movement of kinetochores along microtubules.

Structure and Substrate Recruitment of the Human Spindle Checkpoint Kinase Bub1

In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

Phosphorylation-facilitated sumoylation of MEF2C negatively regulates its transcriptional activity

BackgroundSumoylation has emerged as an important posttranslational regulatory mechanism for transcription factors and cofactors. Sumoylation of many transcription factors represses their transcriptional activities. The myocyte enhancer factor 2 (MEF2) family of transcription factors plays an important role in regulating gene expression during myogenesis and has been recently shown to be sumoylated.ResultsConsistent with earlier reports, we show that sumoylation of MEF2C at K391 inhibits its transcriptional activity. Sumoylation of MEF2C does not block its DNA-binding activity. A small C-terminal fragment of MEF2C containing K391, referred to as delta-N2-MEF2C, is efficiently sumoylated and, when targeted to DNA, represses transcription at neighbouring promoters. Because delta-N2-MEF2C lacks the binding site for class II histone deacetylases (HDACs), this result suggests that sumoylation of MEF2C may help to recruit transcriptional repressors other than these HDACs. Intriguingly, we show that phosphorylation of S396 in MEF2C, a residue in close proximity to the major sumoylation site (K391) and known to be phosphorylated in vivo, enhances sumoylation of delta- N2-MEF2C in vitro. The S396A mutation reduces sumoylation of MEF2C in vivo and enhances the transcription activity of MEF2C in reporter assays.ConclusionWe propose that phosphorylation of MEF2C at S396 facilitates its sumoylation at K391, which in turn recruits yet unidentified co-repressors to inhibit transcription. Our studies further suggest that sumoylation motifs containing a phosphorylated serine or an acidic residue at the +5 position might be more efficiently sumoylated.

Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint

The spindle checkpoint ensures the accuracy of chromosome segregation during mitosis. The protein serine/threonine kinase, Mps1, is a critical component of the spindle checkpoint in human cells and regulates the kinetochore localization of key checkpoint proteins. The kinase activity of Mps1 is required for the spindle checkpoint, but how Mps1 is activated during mitosis is unclear. Here, we show that the endogenous Mps1 in mitotic HeLa cells is phosphorylated on T676, a residue in the activation loop. This phosphorylation event on Mps1 is required for its kinase activity in vitro and for spindle checkpoint signaling in vivo. T676 phosphorylation of Mps1 increases during mitosis and can occur through intermolecular/trans autophosphorylation. Induced dimerization of Mps1 is sufficient to activate its kinase activity in cells. We speculate that the kinetochore localization of Mps1 raises its local concentration, leading to its activation during mitosis through more efficient trans autophosphorylation.

Crm1-Mediated Nuclear Export of Cdc14 is Required for the Completion of Cytokinesis in Budding Yeast

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.

Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells

Human Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion during mitosis. Heterochromatin protein 1 (HP1) has been proposed to recruit Sgo1 to mitotic centromeres. We show that the molecular interaction targeting HP1 to mitotic centromeres is incompatible with HP1 further recruiting Sgo1. Our results clarify the role of centromeric HP1 in chromosome segregation.

Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines

High-content, image-based screens enable the identification of compounds that induce cellular responses similar to those of known drugs but through different chemical structures or targets. A central challenge in designing phenotypic screens is choosing suitable imaging biomarkers. Here we present a method for systematically identifying optimal reporter cell lines for annotating compound libraries (ORACLs), whose phenotypic profiles most accurately classify a training set of known drugs. We generate a library of fluorescently tagged reporter cell lines, and let analytical criteria determine which among them—the ORACL—best classifies compounds into multiple, diverse drug classes. We demonstrate that an ORACL can functionally annotate large compound libraries across diverse drug classes in a single-pass screen and confirm high prediction accuracy by means of orthogonal, secondary validation assays. Our approach will increase the efficiency, scale and accuracy of phenotypic screens by maximizing their discriminatory power.

Kinase signaling in the spindle checkpoint

The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the “wait anaphase” signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects.

Targeting the Spindle Checkpoint in Cancer Chemotherapy

Proper chromosome segregation during mitosis is critical for cells to inherit the correct number of chromosomes and maintain genetic stability. The spindle checkpoint is a cell-cycle surveillance mechanism that prevents premature sister-chromatid separation and ensures the fidelity of chromosome segregation. A defective spindle checkpoint results in aneuploidy and contributes to tumorigenesis. On the other hand, many tumor cells still exhibit a partially functional spindle checkpoint and undergo prolonged mitotic arrest followed by apoptosis when treated with the antimitotic class of anticancer drugs, such as paclitaxel (Taxol). Recent studies have shown that a more complete inactivation of the spindle checkpoint reduces the efficacy of these drugs in eliciting apoptosis in cultured cancer cells. Therefore, quantitative differences in the strength of the spindle checkpoint may influence the efficacy of antimitotic drugs in cancer chemotherapy.