Michael J. Emanuele

A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene

Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS oncogene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we observed a strong enrichment for genes with mitotic functions. We describe a pathway involving the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome that, when inhibited, results in prometaphase accumulation and the subsequent death of Ras mutant cells. Gene expression analysis indicates that reduced expression of genes in this pathway correlates with increased survival of patients bearing tumors with a Ras transcriptional signature. Our results suggest a previously underappreciated role for Ras in mitotic progression and demonstrate a pharmacologically tractable pathway for the potential treatment of cancers harboring Ras mutations.

Global Identification of Modular Cullin-RING Ligase Substrates

Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.

Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly

The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle–dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

Multiple mechanisms of chromosome movement in vertebrate cells mediated through the Ndc80 complex and dynein/dynactin

Kinetochores bind microtubules laterally in a transient fashion and stably, by insertion of plus ends. These pathways may exist to carry out distinct tasks during different stages of mitosis and likely depend on distinct molecular mechanisms. On isolated chromosomes, we found microtubule nucleation/binding depended additively on both dynein/dynactin and on the Ndc80/Hec1 complex. Studying chromosome movement in living Xenopus cells within the simplified geometry of monopolar spindles, we quantified the relative contributions of dynein/dynactin and the Ndc80/Hec1 complex. Inhibition of dynein/dynactin alone had minor effects but did suppress transient, rapid, poleward movements. In contrast, inhibition of the Ndc80 complex blocked normal end-on attachments of microtubules to kinetochores resulting in persistent rapid poleward movements that required dynein/dynactin. In normal cells with bipolar spindles, dynein/dynactin activity on its own allowed attachment and rapid movement of chromosomes on prometaphase spindles but failed to support metaphase alignment and chromatid movement in anaphase. Thus, in prometaphase, dynein/dynactin likely mediates early transient, lateral interactions of kinetochores and microtubules. However, mature attachment via the Ndc80 complex is essential for metaphase alignment and anaphase A.

Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate

The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box–dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.

Xenopus Cep57 is a novel kinetochore component involved in microtubule attachment.

For chromosomes to congress and segregate during cell division, kinetochores must form stable attachments with spindle microtubules. We find that the centrosome protein, xCep57, localizes to kinetochores and interacts with the kinetochore proteins Zwint, Mis12, and CLIP-170. Immunodepletion of xCep57 from egg extracts yields weakened and elongated bipolar spindles which fail to align chromosomes. In the absence of xCep57, tension is lost between sister kinetochores, and spindle microtubules are no longer resistant to low doses of nocodazole. xCep57 inhibition on isolated mitotic chromosomes inhibits kinetochore-microtubule binding in vitro. xCep57 also interacts with gamma-tubulin. In xCep57 immunodepleted extracts, sperm centrosomes nucleate with normal kinetics, but are unable maintain microtubule anchorage. This characterization places xCep57 in a novel class of proteins required for stable microtubule attachments at the kinetochore and at the centrosome and suggests that the mechanism of microtubule binding at these two places is mechanistically similar.

Evolutionarily conserved protein ERH controls CENP-E mRNA splicing and is required for the survival of KRAS mutant cancer cells

Cancers with Ras mutations represent a major therapeutic problem. Recent RNAi screens have uncovered multiple nononcogene addiction pathways that are necessary for the survival of Ras mutant cells. Here, we identify the evolutionarily conserved gene enhancer of rudimentary homolog (ERH), in which depletion causes greater toxicity in cancer cells with mutations in the small GTPase KRAS compared with KRAS WT cells. ERH interacts with the spliceosome protein SNRPD3 and is required for the mRNA splicing of the mitotic motor protein CENP-E. Loss of ERH leads to loss of CENP-E and consequently, chromosome congression defects. Gene expression profiling indicates that ERH is required for the expression of multiple cell cycle genes, and the gene expression signature resulting from ERH down-regulation inversely correlates with KRAS signatures. Clinically, tumor ERH expression is inversely associated with survival of colorectal cancer patients whose tumors harbor KRAS mutations. Together, these findings identify a role of ERH in mRNA splicing and mitosis, and they provide evidence that KRAS mutant cancer cells are dependent on ERH for their survival.

A Hec of a microtubule attachment

The central function of kinetochores is to grasp a dynamic microtubule. Structural, biochemical and cell biological approaches have converged to uncover a microtubule-binding activity within the Ndc80/HEC1 complex, providing a satisfying answer to a question that has puzzled biologists for the last century.

Probing kinetochore structure and function using xenopus laevis frog egg extracts

Kinetochores are multiprotein machines that initiate mitotic checkpoint signaling and control chromosome movement through interactions with microtubules. Our lab has utilized Xenopus laevis frog egg extracts to investigate the requirements for kinetochore assembly and disassembly in vertebrates. Egg extracts support the assembly of functional kinetochores that are capable of binding microtubules, aligning and segregating chromosomes, and sending spindle checkpoint signals. This is the only in vitro system that assembles functional kinetochores, making it particularly well suited for these types of studies. Probing kinetochore assembly using the biochemically tractable egg extract system has elucidated the intricate assembly requirements for numerous vertebrate kinetochore proteins. The following techniques have been used to characterize kinetochore assembly requirements. In addition, we describe assays that we utilized to identify factors that promote maintenance of preassembled kinetochores and those that induce kinetochore disassembly.