Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

Background

Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.

Methodology/Principal Findings

We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.

Conclusions/Significance

These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.     

Focal adhesion kinase plays a dual role in TRAIL resistance and metastatic outgrowth of malignant melanoma

Despite remarkable advances in therapeutic interventions, malignant melanoma (MM) remains a life-threating disease. Following high initial response rates to targeted kinase-inhibition metastases quickly acquire resistance and present with enhanced tumor progression and invasion, demanding alternative treatment options. We show 2^nd generation hexameric TRAIL-receptor-agonist IZI1551 (IZI) to effectively induce apoptosis in MM cells irrespective of the intrinsic BRAF/NRAS mutation status. Conditioning to the EC₅₀ dose of IZI converted the phenotype of IZI-sensitive parental MM cells into a fast proliferating and invasive, IZI-resistant metastasis. Mechanistically, we identified focal adhesion kinase (FAK) to play a dual role in phenotype-switching. In the cytosol, activated FAK triggers survival pathways in a PI3K- and MAPK-dependent manner. In the nucleus, the FERM domain of FAK prevents activation of wtp53, as being expressed in the majority of MM, and consequently intrinsic apoptosis. Caspase-8-mediated cleavage of FAK as well as FAK knockdown, and pharmacological inhibition, respectively, reverted the metastatic phenotype-switch and restored IZI responsiveness. FAK inhibition also re-sensitized MM cells isolated from patient metastasis that had relapsed from targeted kinase inhibition to cell death, irrespective of the intrinsic BRAF/NRAS mutation status. Hence, FAK-inhibition alone or in combination with 2nd generation TRAIL-receptor agonists may be recommended for treatment of initially resistant and relapsed MM, respectively.Subject terms: Metastasis, Target identification     

Blood proteome of acute intracranial hemorrhage in infant victims of abusive head trauma

Abusive head trauma (AHT) is a leading cause of mortality and morbidity in infants. While the reported incidence is close to 40 cases per 100′000 births/year, misdiagnoses are commonly observed in cases with atypical, subacute, or chronic presentation. Currently, standard clinical evaluation of inflicted intracranial hemorrhagic injury (ICH) in infants urgently requires a screening test able to identify infants who need additional investigations. Blood biomarkers characteristic of AHT may assist in detecting these infants, improving prognosis through early medical care. To date, the application of innovative omics technologies in retrospective studies of AHT in infants is rare, due also to the blood serum and cerebrospinal fluid of AHT cases being scarce and not systematically accessible. Here, we explored the circulating blood proteomes of infants with severe AHT and their atraumatic controls. We discovered 165 circulating serum proteins that display differential changes in AHT cases compared with atraumatic controls. The peripheral blood proteomes of pediatric AHT commonly reflect: (i) potentially secreted proteome from injured brain, and (ii) proteome dysregulated in the system's circulation by successive biological events following acute ICH. This study opens up a novel opportunity for research efforts in clinical screening of AHT cases.     

B-RAF mutant alleles associated with Langerhans cell histiocytosis, a granulomatous pediatric disease

Background

Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or ‘LCH cells’. Badalian-Very et al. recently reported the presence of a canonical ^V600EB-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.

Methods and Results

Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using ‘next generation’ pyrosequencing. In 9 cases the mutation identified was ^V600EB-RAF. In 2 cases novel polymorphisms were identified. A somatic ^600DLATB-RAF insertion mimicked the structural and functional consequences of the ^V600EB-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The ^600DLATB-RAF and ^V600EB-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%–2% relative mutation abundance. A novel germ line ^T599AB-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, ^T599AB-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.

Conclusions

Our data confirmed presence of the ^V600EB-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that ^V600EB-RAF and ^600DLATB-RAF mutations are somatic mutants enriched in LCH CD1a⁺ cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line ^T599AB-RAF allele.     

The eroded genome of a Psychotria leaf symbiont: hypotheses about lifestyle and interactions with its plant host

Several plant species of the genus Psychotria (Rubiaceae) harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is also generally described as obligatory because plants devoid of symbionts fail to develop into mature individuals. We sequenced for the first time the genome of the symbiont of Psychotria kirkii in order to shed some light on the nature of their symbiotic relationship. We found that the 4?Mb genome of Candidatus Burkholderia kirkii (B.?kirkii) is small for a Burkholderia species and displays features consistent with ongoing genome erosion such as large proportions of pseudogenes and transposable elements. Reductive genome evolution affected a wide array of functional categories that may hinder the ability of the symbiont to be free-living. The genome does not encode functions commonly found in plant symbionts such as nitrogen fixation or plant hormone metabolism. Instead, a collection of genes for secondary metabolites' synthesis is located on the 140?kb plasmid of B.?kirkii and suggests that leaf nodule symbiosis benefits the host by providing protection against herbivores or pathogens.     

APDB: a novel measure for benchmarking sequence alignment methods without reference alignments

MOTIVATION: We describe APDB, a novel measure for evaluating the quality of a protein sequence alignment, given two or more PDB structures. This evaluation does not require a reference alignment or a structure superposition. APDB is designed to efficiently and objectively benchmark multiple sequence alignment methods. RESULTS: Using existing collections of reference multiple sequence alignments and existing alignment methods, we show that APDB gives results that are consistent with those obtained using conventional evaluations. We also show that APDB is suitable for evaluating sequence alignments that are structurally equivalent. We conclude that APDB provides an alternative to more conventional methods used for benchmarking sequence alignment packages.     

Molecular characterization of a series of 990 index patients with albinism

Albinism is a clinically and genetically heterogeneous disease characterized by variable degrees of hypopigmentation and by nystagmus, foveal hypoplasia, and chiasmatic misrouting of the optic nerves. The wide phenotypic heterogeneity impedes the establishment of phenotype–genotype correlations. To obtain a precise diagnosis, we screened the 19 known albinism genes in 990 index patients using targeted next‐generation sequencing (NGS) and high‐resolution comparative genomic hybridization. A molecular diagnosis was obtained in 72.32% of patients. A total of 243 new pathogenic variants were identified. Intragenic rearrangements represented 10.8% of all pathogenic alleles. NGS panel analysis allowed establishing a diagnosis for the rarest forms of the disease, which could not be diagnosed otherwise. Because of the clinical overlap between the different forms of the disease, diagnosis nowadays clearly relies on molecular grounds.