Non small cell lung cancer H460 clones exhibit a high degree of heterogeneity in signaling states.Clones with similar patterns of basal signaling heterogeneity have similar paclitaxel sensitivities.Models of signaling heterogeneity among the clones can be used to classify sensitivity to paclitaxel for other cancer populations.

Rationale Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells (ECs), apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 is required for apical junction clearance, as well as for regulation of Rho GTPase activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis. Objective The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis. Methods and Results Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of EC apical junctions during vascular tubulogenesis depends on Ras interacting protein 1 (Rasip1), as well as the GTPase Cdc42 and the kinase Pak4. Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all block EC tubulogenesis. By contrast, inactivation of RhoA signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII either prior to, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles. Conclusions Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.

High throughput screening of insulin secretion is intractable with current methods. We developed a secreted insulin-luciferase system (Ins-GLuc) in β cells that is rapid, inexpensive, and amenable to 96- and 384-well formats. We treated stable Ins-GLuc-expressing MIN6 cells overnight with 6298 marine natural product fractions. The cells were then washed to remove media and chemicals, followed by stimulation with glucose in the diazoxide paradigm. These conditions allowed the discovery of many insulin secretion suppressors and potentiators. The mechanisms of action of these natural products must be long-lasting given the continuance of secretory phenotypes in the absence of chemical treatment. We anticipate that these natural products and their target pathways will lead to a greater understanding of glucose-stimulated insulin secretion. Keywords: insulin, Gaussia, high-throughput screening, pancreatic islet beta cell, marine natural products.

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.

The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertensionrelated disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNAmediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phosphatidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phosphorylation, and activation of the kinase upstream of ULK1, the AMPactivated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related proline-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or -starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.T he with-no-lysine (K) (WNK) protein kinase family is an evolutionarily conserved, atypical group of serine/threonine kinases with the conserved ATP-binding lysine residue shifted to a different position within the kinase domain (1, 2). WNKs are the only kinases in the eukaryotic protein kinase superfamily with this unusual arrangement. This arrangement confers on them unique structural and functional properties (3). There are four WNK proteins in mammals, of which WNK1 is the largest, over 2,000 residues, and most widely expressed (4).The best-characterized function of WNKs is their binding and activation of downstream target kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK) (5-7). Once activated, OSR1 and SPAK phosphorylate and regulate downstream cation-chloride cotransporters of the SLC12 family (8-10). This WNK-SPAK/OSR1 pathway enables cells to adjust intracellular ions and cell volume in response to ion imbalances and osmotic stress (11). It is noteworthy that mutations in the regulatory components of the WNK pathway, including WNK1, WNK4, kelch-likes (KLHLs), and cullins, have been shown to cause increased expression of WNKs and ion reabsorption defects in kidney that lead to hypertension-related genetic diseases, such as Gordon's syndrome (pseudohypoaldosteronism II) (12-16). In addition, WNKs have been linked to the regulation of cell proliferation (17, 18), cell death (19), cell migration (20-23), endocytosis (24-27...

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

A fundamental challenge in treating disease is identifying molecular states that affect cellular responses to drugs. Here, we focus on GSK-3, a key regulator for many of the hallmark behaviors of cancer cells. We alter GSK-3 activity in colon epithelial cells to test its role in modulating drug response. We find that GSK-3 activity broadly affects the cellular sensitivities to a panel of oncology drugs and kinase inhibitors. Specifically, inhibition of GSK-3 activity can strongly desensitize or sensitize cells to kinase inhibitors (e.g. mTOR or PLK1 inhibitors, respectively). Additionally, colorectal cancer cell lines, in which GSK-3 function is commonly suppressed, are resistant to mTOR inhibitors and yet highly sensitive to PLK1 inhibitors and this is further exacerbated by additional GSK-3 inhibition. Finally, by conducting a kinome-wide RNAi screen, we find that GSK-3 modulates the cell proliferative phenotype of a significant fraction (~35%) of the kinome, which includes ~50% of current, clinically relevant kinase-targeted drugs. Our results highlight an under-appreciated interplay of GSK-3 with therapeutically important kinases and suggest strategies for identifying disease-specific molecular profiles that can guide optimal selection of drug treatment.

Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions.