Structural studies of cerebral cavernous malformations 2 (CCM2) reveal a folded helical domain at its C-terminus

AbstractCerebral cavernous malformations (CCM) are neurovascular dysplasias affecting up to 0.5% of the population. Mutations in the CCM2 gene are associated with acquisition of CCM. We identify a previously uncharacterized domain at the C-terminus of CCM2 and determine its 1.9Å resolution crystal structure. Because this domain is structurally homologous to the N-terminal domain of harmonin, we name it the CCM2 harmonin-homology domain or HHD. CCM2 HHD is observed in two conformations, and we employ analytical ultracentrifugation to test its oligomerization. Additionally, CCM2 HHD contains an unusually long 13-residue 310 helix. This study provides the first structural characterization of CCM2.Structured summary of protein interactionsCCM2 binds to CCM3 by pull down (View interaction)CCM2 and CCM2 bind by X-ray crystallography (View interaction)CCM2 and CCM2 bind by molecular sieving (View interaction

Molecular Basis for Integrin Adhesion Receptor Binding to p21-activated kinase 4 (PAK4)

Integrin adhesion receptors provide links between extracellular ligands and cytoplasmic signaling. Multiple kinases have been found to directly engage with integrin β tails, but the molecular basis for these interactions remain unknown. Here, we assess the interaction between the kinase domain of p21-activated kinase 4 (PAK4) and the cytoplasmic tail of integrin β5. We determine three crystal structures of PAK4-β5 integrin complexes and identify the PAK-binding site. This is a region in the membrane-proximal half of the β5 tail and confirmed by site-directed mutagenesis. The β5 tail engages the kinase substrate-binding groove and positions the non-phosphorylatable integrin residue Glu767 at the phosphoacceptor site. Consistent with this, integrin β5 is poorly phosphorylated by PAK4, and in keeping with its ability to occlude the substrate-binding site, weakly inhibits kinase activity. These findings demonstrate the molecular basis for β5 integrin-PAK4 interactions but suggest modifications in understanding the potential cellular role of this interaction

BIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR Mutations

Background: Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process. Methods and Findings: To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis. Conclusions: Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFRmutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinibinduced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors

Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles

SummaryMutations in the juxtamembrane and kinase domains of FLT3 are common in AML, but it is not known whether alterations outside these regions contribute to leukemogenesis. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in AML patients without known FLT3 mutations and experimentally tested the consequences of each candidate leukemogenic allele. This approach identified gain-of-function mutations that activated downstream signaling and conferred sensitivity to FLT3 inhibition and alleles that were not associated with kinase activation, including mutations in the catalytic domain. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation and underscore the importance of functional studies to distinguish “driver” mutations underlying tumorigenesis from biologically neutral “passenger” alterations

Data Publication with the Structural Biology Data Grid Supports Live Analysis

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis

Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis

SummaryPolycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are clonal disorders arising from hematopoietic progenitors. An internet-based protocol was used to collect clinical information and biological specimens from patients with these diseases. High-throughput DNA resequencing identified a recurrent somatic missense mutation JAK2V617F in granulocyte DNA samples of 121 of 164 PV patients, of which 41 had homozygous and 80 had heterozygous mutations. Molecular and cytogenetic analyses demonstrated that homozygous mutations were due to duplication of the mutant allele. JAK2V617F was also identified in granulocyte DNA samples from 37 of 115 ET and 16 of 46 MMM patients, but was not observed in 269 normal individuals. In vitro analysis demonstrated that JAK2V617F is a constitutively active tyrosine kinase