The innate immune signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes ϳ75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo.Vaults are predominantly cytoplasmic ribonucleoprotein particles that have been conserved throughout evolution and are found in phylogenies as diverse as those of mammals, avians, amphibians, sea urchins, and slime molds (1). Many different roles including nucleocytoplasmic transport have been proposed for vaults since their first description in 1986 (2). However, their normal cellular function remains nebulous. Mammalian vaults comprise three proteins, the major vault protein (MVP) 1 (3), the vault poly(A)DP-ribose polymerase (VPARP) (4), and the telomerase-associated protein 1 (TEP1) (5) and one or more small untranslated RNAs (6). Vaults have been implicated in the phenomenon of multidrug resistance and as prognostic markers for cancer chemotherapy failure (7,8). One recent study has shown that the major vault protein is involved directly in the efflux of drugs from the nucleus (9). Although the majority of vaults are found in the cytoplasm, small amounts have been localized to the nuclear pore complexes (10). Recently a 31-Å resolution structure of the vault has been published indicating that vaults have a hollow interior consistent with a transport or sequestration function. Scanning transmission electron microscopic analysis has shown that the molecular mass of the vault is 12.9 Ϯ 1 MDa, and cryo-EM single-particle reconstruction has provided overall dimensions of 42 ϫ 75 nm (11). Freeze-etch images of the vault on polylysine-coated mica show that each half of the vault midsection can open into eight distinct "petals" (3), which has lead to the proposal that vaults may open and close in vivo. The MVP is presumed to be present in 96 copies/vault, based on the observed symmetry of the particle and the estimate that MVP accounts for ϳ75% of the total protein mass in the particle. In many way...

Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase alpha from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome-editing tools. Recently, conditional reprogramming of cells (CRC) with a Rho-associated protein kinase (ROCK) inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the life span of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial cell growth medium, instead of F medium, and 2% O, instead of 21% O, that extend HBEC life span while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell, and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof-of-concept, CRISPR/Cas9 genome editing and cloning were used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

Cells that suffer substantial inhibition of DNA replication halt their cell cycle via a checkpoint response mediated by the PI3 kinases ATM and ATR. It is unclear how cells cope with milder replication insults, which are under the threshold for ATM and ATR activation. A third PI3 kinase, DNA-dependent protein kinase (DNA-PK), is also activated following replication inhibition, but the role DNA-PK might play in response to perturbed replication is unclear, since this kinase does not activate the signaling cascades involved in the S-phase checkpoint. Here we report that mild, transient drug-induced perturbation of DNA replication rapidly induced DNA breaks that promptly disappeared in cells that contained a functional DNA-PK whereas such breaks persisted in cells that were deficient in DNA-PK activity. After the initial transient burst of DNA breaks, cells with a functional DNA-PK did not halt replication and continued to synthesize DNA at a slow pace in the presence of replication inhibitors. In contrast, DNA-PK deficient cells subject to low levels of replication inhibition halted cell cycle progression via an ATR-mediated S-phase checkpoint. The ATM kinase was dispensable for the induction of the initial DNA breaks. These observations suggest that DNA-PK is involved in setting a high threshold for the ATR-Chk1-mediated S-phase checkpoint by promptly repairing DNA breaks that appear immediately following inhibition of DNA replication.