Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a ...bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + β fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.
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▸ The c-di-GMP binding domain (CBD) of STING is a wing-shaped dimer with a novel fold ▸ c-di-GMP assumes a symmetrical conformation and binds at the STING dimer interface ▸ STING is autoinhibited by an intramolecular interaction which c-di-GMP antagonizes
RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival
. RAF activity is tightly ...regulated and inappropriate activation is a frequent cause of cancer
; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of ...growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway). Expected final online publication date for the
, Volume 93 is June 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Mutant selective irreversible pyrimidine-based EGFR kinase inhibitors, including WZ4002, CO-1686, and AZD9291, are effective in preclinical models and in lung cancer patients harboring the EGFR T790M ...gefitinib/erlotinib resistance mutation. However, little is known about how cancers develop acquired resistance to this class of EGFR inhibitors. We sought to identify and study EGFR mutations that confer resistance to this class of agents.
We performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in EGFR-mutant (sensitizing alone or with concurrent EGFR T790M) Ba/F3 cells and selected drug-resistant clones. We evaluated the sensitivity of EGFR inhibitors in models harboring drug-resistant EGFR mutations.
We identified 3 major drug resistance mutations. EGFR L718Q, L844V, and C797S cause resistance to both WZ4002 and CO-1686 while, in contrast, only EGFR C797S leads to AZD9291 resistance. Cells containing an EGFR-sensitizing mutation, Del 19 or L858R, in conjunction with L718Q, L844V, or C797S retain sensitivity to quinazoline-based EGFR inhibitors, gefitinib and afatinib. The C797S mutation, in the presence of Del 19 or L858R and T790M, causes resistance to all current EGFR inhibitors, but L858R/T790M/C797S remains partially sensitive to cetuximab which leads to disruption of EGFR dimerization.
Our findings provide insights into resistance mechanisms to irreversible pyrimidine-based EGFR inhibitors and identify specific genomic contexts in which sensitivity is retained to existing clinical EGFR inhibitors. These findings will guide the development of new strategies to inhibit EGFR.
Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain ...proteins associate with a variety of other cellular factors and directly nucleate actin polymerization through a novel mechanism. The signature formin homology 2 (FH2) domain initiates filament assembly and remains persistently associated with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed formins in animals and fungi are autoinhibited through intramolecular interactions and appear to be activated by Rho GTPases and additional factors. Other classes of formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.
Recently launched multichannel geostationary Earth orbit (GEO) satellite
sensors, such as the Geostationary Ocean Color Imager (GOCI) and the Advanced
Himawari Imager (AHI), provide aerosol products ...over East Asia with high
accuracy, which enables the monitoring of rapid diurnal variations and the
transboundary transport of aerosols. Most aerosol studies to date have used
low Earth orbit (LEO) satellite sensors, such as the Moderate Resolution
Imaging Spectroradiometer (MODIS) and the Multi-angle Imaging
Spectroradiometer (MISR), with a maximum of one or two overpass daylight
times per day from midlatitudes to low latitudes. Thus, the demand for new GEO
observations with high temporal resolution and improved accuracy has been
significant. In this study the latest versions of aerosol optical depth
(AOD) products from three LEO sensors – MODIS (Dark Target, Deep Blue, and
MAIAC), MISR, and the Visible/Infrared Imager Radiometer Suite
(VIIRS), along with two GEO sensors (GOCI and AHI), are validated,
compared, and integrated for a period during the Korea–United States Air
Quality Study (KORUS-AQ) field campaign from 1 May to 12 June 2016 over East
Asia. The AOD products analyzed here generally have high accuracy with high
R (0.84–0.93) and low RMSE (0.12–0.17), but their error characteristics differ
according to the use of several different surface-reflectance estimation
methods. High-accuracy near-real-time GOCI and AHI measurements facilitate
the detection of rapid AOD changes, such as smoke aerosol transport from
Russia to Japan on 18–21 May 2016, heavy pollution transport from China to
the Korean Peninsula on 25 May 2016, and local emission transport from the Seoul
Metropolitan Area to the Yellow Sea in South Korea on 5 June 2016. These
high-temporal-resolution GEO measurements result in more representative
daily AOD values and make a greater contribution to a combined daily AOD
product assembled by median value selection with a 0.5∘×0.5∘ grid resolution. The combined AOD is spatially continuous and has
a greater number of pixels with high accuracy (fraction within expected
error range of 0.61) than individual products. This study characterizes
aerosol measurements from LEO and GEO satellites currently in operation over
East Asia, and the results presented here can be used to evaluate satellite
measurement bias and air quality models.
Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these ...agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the "gatekeeper" residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a "generic" resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.
Focal adhesion kinase (FAK) is a scaffold and tyrosine kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) domain. Recent structural ...work reveals that regulatory protein partners convert auto-inhibited FAK into its active state by binding to its FERM domain. Further, the identity of FAK FERM domain-interacting proteins yields clues as to how FAK coordinates diverse cellular responses, including cell adhesion, polarization, migration, survival and death, and suggests that FERM domains might mediate information transfer between the cell cortex and nucleus. Importantly, the FAK FERM domain might act as a paradigm for the actions of other FERM domain-containing proteins.
EGFR and other ErbB-family tyrosine kinases are overexpressed in many human tumors, and their aberrant expression and mutational activation is associated with the development, progression and ...aggressiveness of a number of malignancies. Thus the EGFR kinase has long been recognized as a potential drug target in oncology, and small-molecule inhibitors have been under development for more than two decades. As a result of their effectiveness in treating non-small cell lung cancers (NSCLCs) driven by somatic mutations in the EGFR kinase, gefitinib and erlotinib were the first EGFR tyrosine kinase inhibitors (TKIs) approved for clinical use. Ironically, these drugs found their target against mutant forms of the EGFR kinase, which have altered enzyme active sites, and not against the wild type (WT) kinase against which their potency and selectivity was carefully honed. Here we review recent structural and enzymological studies that explore the exquisite sensitivity of a subset of these lung cancer mutants to gefitinib and erlotinib. We discuss available structural evidence for the mechanisms of activation of the EGFR kinase by these mutants, and compare it to physiologic activation of the kinase by ligand-induced dimerization. Finally, we consider the mechanisms by which the secondary T790M “gatekeeper” mutation confers resistance to gefitinib and erlotinib.
Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from ...integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation.
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