Ras is frequently mutated in cancer; however, there is a lack of consensus in the literature regarding the cancer mutation frequency of Ras, with quoted values varying from 10-30%. This variability is at least in part due to the selective aggregation of data from different databases and the dominant influence of particular cancer types and particular Ras isoforms within these datasets. In order to provide a more definitive figure for Ras mutation frequency in cancer, we cross-referenced the data in all major publicly accessible cancer mutation databases to determine reliable mutation frequency values for each Ras isoform in all major cancer types. These percentages were then applied to current US cancer incidence statistics to estimate the number of new patients each year that have Ras-mutant cancers. We find that ~19% of cancer patients harbor Ras mutations; equivalent to ~3.4 million new cases per year worldwide. We discuss the Ras isoform and mutation-specific trends evident within the datasets that are relevant to current Ras-targeted therapies. Research.

Kinetochore fibres (K-fibres) of the spindle apparatus move chromosomes during mitosis. These fibres are discrete bundles of parallel microtubules (MTs) that are crosslinked by inter-MT 'bridges' that are thought to improve fibre stability during chromosomal movement. The identity of these bridges is unknown. Clathrin is a multimeric protein that has been shown to stabilise K-fibres during early mitosis by a mechanism independent of its role in membrane trafficking. In this study, we show that clathrin at the mitotic spindle is in a transforming acidic colied-coil protein 3 (TACC3)/colonic, hepatic tumour overexpressed gene (ch-TOG)/clathrin complex. The complex is anchored to the spindle by TACC3 and ch-TOG. Ultrastructural analysis of clathrin-depleted K-fibres revealed a selective loss of a population of short inter-MT bridges and a general loss of MTs. A similar loss of short inter-MT bridges was observed in TACC3-depleted K-fibres. Finally, immunogold labelling confirmed that inter-MT bridges in K-fibres contain clathrin. Our results suggest that the TACC3/ch-TOG/clathrin complex is an inter-MT bridge that stabilises K-fibres by physical crosslinking and by reducing rates of MT catastrophe.

Aurora A phosphorylation-induced interaction of TACC3 and clathrin coordinates adjacent domains in each protein to create a microtubule-binding interface, whereas a distinct site in TACC3 recruits ch-TOG to mitotic spindles.

The human protein kinome comprises 535 proteins that, with the exception of approximately 50 pseudokinases, control intracellular signaling networks by catalyzing the phosphorylation of multiple protein substrates. While a major research focus of the last 30 years has been cancer-associated Tyr and Ser/Thr kinases, over 85% of the kinome has been identified to be dysregulated in at least one disease or developmental disorder. Despite this remarkable statistic, for the majority of protein kinases and pseudokinases, there are currently no inhibitors progressing toward the clinic, and in most cases, details of their physiologic and pathologic mechanisms remain at least partially obscure. By curating and annotating data from the literature and major public databases of phosphorylation sites, kinases, and disease associations, we generate an unbiased resource that highlights areas of unmet need within the kinome. We discuss strategies and challenges associated with characterizing catalytic and noncatalytic outputs in cells, and describe successes and new frontiers that will support more comprehensive cancer-targeting and therapeutic evaluation in the future. .

The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.

Kinetochore fibers (K-fibers) of the mitotic spindle are force-generating units that power chromosome movement during mitosis. K-fibers are composed of many microtubules that are held together throughout their length. Here, we show, using 3D electron microscopy, that K-fiber microtubules (MTs) are connected by a network of MT connectors. We term this network ‘the mesh’. The K-fiber mesh is made of linked multipolar connectors. Each connector has up to four struts, so that a single connector can link up to four MTs. Molecular manipulation of the mesh by overexpression of TACC3 causes disorganization of the K-fiber MTs. Optimal stabilization of K-fibers by the mesh is required for normal progression through mitosis. We propose that the mesh stabilizes K-fibers by pulling MTs together and thereby maintaining the integrity of the fiber. Our work thus identifies the K-fiber meshwork of linked multipolar connectors as a key integrator and determinant of K-fiber structure and function.DOI: http://dx.doi.org/10.7554/eLife.07635.001

Hsp72 is a novel mitotic substrate of Nek6, and, together, these proteins play an essential role in assembly of robust mitotic spindles capable of efficient chromosome congression through K-fiber recruitment of the ch-TOG and TACC3 complex.

Clathrin is crucial for endocytosis and plays a recently described role in mitosis. Two clathrin heavy chains (CHCs) are found in humans: the ubiquitous CHC17 and CHC22, a CHC that is enriched in skeletal muscle. Functional differences have been proposed for these clathrins despite high sequence homology. Here, we compared each paralogue in functional assays of endocytosis and mitosis. We find that CHC17 and CHC22 are functionally equivalent. We also describe how previous work on CHC22 has involved a splice variant that is not usually expressed in cells.