Systematic phosphorylation analysis of human mitotic protein complexes.

Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1

The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion.

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2-7 subcomplex of the replicative Cdc45-MCM-GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively

Systematic analysis of human protein complexes identifies chromosome segregation proteins.

Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells

Control of Drosophila endocycles by E2F and CRL4(CDT2)

Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis1,2. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world’s biomass3,4. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase5,6. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modelling, we show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4CDT2 ubiquitin ligase, and this in turn mediates the destruction of E2F1 (ref. 7). We propose that it is the transient loss of E2F1 during S phases that creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, we find that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants1,2,8, indicating that elements of this mechanism act in most growth-dependent cell cycles

Systematic analysis of human protein complexes identifies chromosome segregation proteins.

Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells

Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma

The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies

Identifiers for the 21st century: How to design, provision, and reuse persistent identifiers to maximize utility and impact of life science data.

In many disciplines, data are highly decentralized across thousands of online databases (repositories, registries, and knowledgebases). Wringing value from such databases depends on the discipline of data science and on the humble bricks and mortar that make integration possible; identifiers are a core component of this integration infrastructure. Drawing on our experience and on work by other groups, we outline 10 lessons we have learned about the identifier qualities and best practices that facilitate large-scale data integration. Specifically, we propose actions that identifier practitioners (database providers) should take in the design, provision and reuse of identifiers. We also outline the important considerations for those referencing identifiers in various circumstances, including by authors and data generators. While the importance and relevance of each lesson will vary by context, there is a need for increased awareness about how to avoid and manage common identifier problems, especially those related to persistence and web-accessibility/resolvability. We focus strongly on web-based identifiers in the life sciences; however, the principles are broadly relevant to other disciplines

REMBI: Recommended Metadata for Biological Images—enabling reuse of microscopy data in biology

Bioimaging data have significant potential for reuse, but unlocking this potential requires systematic archiving of data and metadata in public databases. We propose draft metadata guidelines to begin addressing the needs of diverse communities within light and electron microscopy. We hope this publication and the proposed Recommended Metadata for Biological Images (REMBI) will stimulate discussions about their implementation and future extension