The Hsp70-Hsp90 go-between Hop/Stip1/Sti1 is a proteostatic switch and may be a drug target in cancer and neurodegeneration.

The Hsp70 and Hsp90 molecular chaperone systems are critical regulators of protein homeostasis (proteostasis) in eukaryotes under normal and stressed conditions. The Hsp70 and Hsp90 systems physically and functionally interact to ensure cellular proteostasis. Co-chaperones interact with Hsp70 and Hsp90 to regulate and to promote their molecular chaperone functions. Mammalian Hop, also called Stip1, and its budding yeast ortholog Sti1 are eukaryote-specific co-chaperones, which have been thought to be essential for substrate ("client") transfer from Hsp70 to Hsp90. Substrate transfer is facilitated by the ability of Hop to interact simultaneously with Hsp70 and Hsp90 as part of a ternary complex. Intriguingly, in prokaryotes, which lack a Hop ortholog, the Hsp70 and Hsp90 orthologs interact directly. Recent evidence shows that eukaryotic Hsp70 and Hsp90 can also form a prokaryote-like binary chaperone complex in the absence of Hop, and that this binary complex displays enhanced protein folding and anti-aggregation activities. The canonical Hsp70-Hop-Hsp90 ternary chaperone complex is essential for optimal maturation and stability of a small subset of clients, including the glucocorticoid receptor, the tyrosine kinase v-Src, and the 26S/30S proteasome. Whereas many cancers have increased levels of Hop, the levels of Hop decrease in the aging human brain. Since Hop is not essential in all eukaryotic cells and organisms, tuning Hop levels or activity might be beneficial for the treatment of cancer and neurodegeneration.

Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles.

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.

Interaction between Ras and Src clones causes interdependent tumor malignancy via Notch signaling in Drosophila.

Cancer tissue often comprises multiple tumor clones with distinct oncogenic alterations such as Ras or Src activation, yet the mechanism by which tumor heterogeneity drives cancer progression remains elusive. Here, we show in Drosophila imaginal epithelium that clones of Ras- or Src-activated benign tumors interact with each other to mutually promote tumor malignancy. Mechanistically, Ras-activated cells upregulate the cell-surface ligand Delta while Src-activated cells upregulate its receptor Notch, leading to Notch activation in Src cells. Elevated Notch signaling induces the transcriptional repressor Zfh1/ZEB1, which downregulates E-cadherin and cell death gene hid, leading to Src-activated invasive tumors. Simultaneously, Notch activation in Src cells upregulates the cytokine Unpaired/IL-6, which activates JAK-STAT signaling in neighboring Ras cells. Elevated JAK-STAT signaling upregulates the BTB-zinc-finger protein Chinmo, which downregulates E-cadherin and thus generates Ras-activated invasive tumors. Our findings provide a mechanistic explanation for how tumor heterogeneity triggers tumor progression via cell-cell interactions.

Zinc transporter SLC39A13/ZIP13 facilitates the metastasis of human ovarian cancer cells via activating Src/FAK signaling pathway.

BACKGROUND: Zinc transporters have been found to be associated with the pathogenesis of numerous human diseases including cancer. As the most lethal gynecologic malignancy, ovarian cancer is characterized by rapid progression and widespread metastases. However, the function and underlying mechanism of zinc transporters in ovarian cancer metastasis remain unclear. METHODS: The relationship between zinc transporter gene expressions and clinical outcomes of ovarian cancer was assessed with the online database Kaplan-Meier plotter ( http://kmplot.com/analysis/ ). Immunohistochemistry was performed to investigate the prognostic importance of ZIP13. The expression of ZIP13 in ovarian cancer cell lines was depleted to explore its effect on proliferation, adhesion, migration, and invasion both in vitro and in vivo assays. RNA-Seq, quantitative RT-PCR, and western blot analysis were performed to explore ZIP13-regulated downstream target genes. RESULTS: The expressions of several zinc transporters were highly associated the clinical outcomes of ovarian cancer patients. Among them, high ZIP13 expression was an independent prognostic factor for poor survival in patients with ovarian cancer. ZIP13 knockout suppressed the malignant phenotypes of ovarian cancer cells both in vitro and in vivo. Further investigation revealed that ZIP13 regulated intracellular zinc distribution and then affected the expressions of genes involved in extracellular matrix organization and cytokine-mediated signaling pathway. This led to the activation of Src/FAK pathway with increased expressions of pro-metastatic genes but decreased expressions of tumor suppressor genes. CONCLUSIONS: ZIP13 is shown to be a novel driver of metastatic progression by modulating the Src/FAK signaling pathway, which may serve as a promising biomarker for prognostic evaluation and targeted therapy in ovarian cancer.

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division.

v-Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v-Src-mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities-mediated chromosome instability. v-Src directly phosphorylates Tyr-15 of cyclin-dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v-Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v-Src restores cancer cell viability reduced by various microtubule-targeting agents (MTAs), although v-Src does not alter cytotoxicity of DNA-damaging anticancer drugs. v-Src causes mitotic slippage of MTAs-treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v-Src also restores cell viability reduced by a polo-like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v-Src. These results suggest that the v-Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src-induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.

Tyrosine-phosphorylation and activation of glucosylceramide synthase by v-Src: Its role in survival of HeLa cells against ceramide.

Sphingolipids represent a family of cellular lipid-molecules that regulate physiological and pathophysiological processes. Glucosylceramide (GlcCer), the simplest glycosphingolipid (GSL), is synthesized from ceramide and UDP-glucose by GlcCer synthase (GCS). Both GlcCer (and resulting GSLs) and ceramide regulate various cellular functions including cell death and multiple drug resistance. Src family tyrosine kinases are up-regulated in various human cancer cells. We examined the effect of v-Src expression on GCS activity, the formation of 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled GlcCer from NBD-ceramide, and the effect of tyrosine132 mutation in GCS on ceramide-induced cytotoxicity in HeLa cells. Expression of v-Src increased the formation of NBD-GlcCer in both intact cells without marked changes in other sphingolipid metabolites and cell homogenates without changing affinities of NBD-ceramide and UDP-glucose. Expression of v-Src also increased tyrosine-phosphorylated levels in GCS proteins in HeLa and HEK293T cells. In HEK293T cells transiently expressing the GCS mutant, GCS-Y132F-HA, showing replacement of the tyrosine132 residue with phenylalanine, tyrosine-phosphorylated levels in GCS proteins were significantly lower than those in control cells expressing the GCS-wild-type-HA. The formation of NBD-GlcCer in HeLa cells stably expressing GCS-Y132F-HA was significantly lower than that in the control. Ceramide-induced cytotoxicity in HeLa-GCS-Y132F-HA cells was significantly greater than in the control. In this study, we showed for the first time that expression of v-Src up-regulated GCS activity via tyrosine phosphorylation of the enzyme in a post-translational manner. Mechanisms of Src-induced resistance to ceramide-induced cytotoxicity are discussed in relation to the Src-induced up-regulation of GCS activity.

The tumor suppressor LATS2 reduces v-Src-induced membrane blebs in a kinase activity-independent manner.

When cells with excess DNA, such as tetraploid cells, undergo cell division, it can contribute to cellular transformation via asymmetrical chromosome segregation-generated genetic diversity. Cell cycle progression of tetraploid cells is suppressed by large tumor suppressor 2 (LATS2) kinase-induced inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP). We recently reported that the oncogene v-Src induces tetraploidy and promotes cell cycle progression of tetraploid cells by suppressing LATS2 activity. We explore here the mechanism by which v-Src suppresses LATS2 activity and the role of LATS2 in v-Src-expressing cells. LATS2 was directly phosphorylated by v-Src and the proto-oncogene c-Src, resulting in decreased LATS2 kinase activity. This kinase-deficient LATS2 accumulated in a YAP transcriptional activity-dependent manner, and knockdown of either LATS2 or the LATS2-binding partner moesin-ezrin-radixin-like protein (Merlin) accelerated v-Src-induced membrane bleb formation. Upon v-Src expression, the interaction of Merlin with LATS2 was increased possibly due to a decrease in Merlin phosphorylation at Ser518, the dephosphorylation of which is required for the open conformation of Merlin and interaction with LATS2. LATS2 was colocalized with Merlin at the plasma membrane in a manner that depends on the Merlin-binding region of LATS2. The bleb formation in v-Src-expressing and LATS2-knockdown cells was rescued by the reexpression of wild-type or kinase-dead LATS2 but not the LATS2 mutant lacking the Merlin-binding region. These results suggest that the kinase-deficient LATS2 plays a role with Merlin at the plasma membrane in the maintenance of cortical rigidity in v-Src-expressing cells, which may cause tumor suppression.

The Anti-Cancer Effect of Linusorb B3 from Flaxseed Oil through the Promotion of Apoptosis, Inhibition of Actin Polymerization, and Suppression of Src Activity in Glioblastoma Cells.

Linusorbs (LOs) are natural peptides found in flaxseed oil that exert various biological activities. Of LOs, LOB3 ([1-9-NαC]-linusorb B3) was reported to have antioxidative and anti-inflammatory activities; however, its anti-cancer activity has been poorly understood. Therefore, this study investigated the anti-cancer effect of LOB3 and its underlying mechanism in glioblastoma cells. LOB3 induced apoptosis and suppressed the proliferation of C6 cells by inhibiting the expression of anti-apoptotic genes, B cell lymphoma 2 (Bcl-2) and p53, as well as promoting the activation of pro-apoptotic caspases, caspase-3 and -9. LOB3 also retarded the migration of C6 cells, which was achieved by suppressing the formation of the actin cytoskeleton critical for the progression, invasion, and metastasis of cancer. Moreover, LOB3 inhibited the activation of the proto-oncogene, Src, and the downstream effector, signal transducer and activator of transcription 3 (STAT3), in C6 cells. Taken together, these results suggest that LOB3 plays an anti-cancer role by inducing apoptosis and inhibiting the migration of C6 cells through the regulation of apoptosis-related molecules, actin polymerization, and proto-oncogenes.

Knockdown of TRPM7 prevents tumor growth, migration, and invasion through the Src, Akt, and JNK pathway in bladder cancer.

BACKGROUND: Bladder cancer (BC) is one of the most common malignancies of the urinary tract. The role of transient receptor potential melastatin 7 (TRPM7) in BC remains unclear. The aim of this study was to investigate the function and signal transduction pathway of TRPM7 in BC. METHODS: T24 and UMUC3 cells were used to evaluate the molecular mechanism of TRPM7 by immunoblot analysis. Small interfering RNA was used to knockdown TRPM7, and the effect of silencing TRPM7 was studied by wound healing, migration, and invasion assays in T24 and UMUC3 cells. Xenograft model study was obtained to analyze the effect of TRPM7 inhibition in vivo. RESULTS: Silencing of TRPM7 decreased the migration and invasion ability of T24 and UMUC3 cells. The phosphorylation of Src, Akt, and JNK (c-Jun N-terminal kinase) was also suppressed by TRPM7 silencing. Src, Akt, and JNK inhibitors effectively inhibited the migration and invasion of T24 and UMUC3 cells. In addition, the TRPM7 inhibitor, carvacrol, limited the tumor size in a xenograft model. CONCLUSION: Our data reveal that TRPM7 regulates the migration and invasion of T24 and UMUC3 cells via the Src, Akt, and JNK signaling pathway. Therefore, TRPM7 suppression could be a potential treatment for BC patients.

Histamine H4 receptor agonists induce epithelial-mesenchymal transition events and enhance mammosphere formation via Src and TGF-ß signaling in breast cancer cells.

Epithelial-mesenchymal transition (EMT) contributes to cell invasion and metastasis during the progression of epithelial cancers. Though preclinical evidence suggests a role for histamine H4 receptor (H4R) in breast cancer growth, its function in the EMT is less known. In this study we proposed to investigate the effects of H4R ligands on EMT and mammosphere formation as a surrogate assay for cancer stem cells in breast cancer cells with different invasive phenotype. We also investigated the participation of Src and TGF-ß signaling in these events. Breast cancer cells were treated with the H4R agonists Clobenpropit, VUF8430 and JNJ28610244 and the H4R antagonist JNJ7777120. Immunodetection studies showed cytoplasmic E-cadherin, cytoplasmic and nuclear beta-catenin, nuclear Slug and an increase in vimentin and α-smooth muscle actin expression. There was also an enhancement in cell migration and invasion assessed by transwell units. All these effects were prevented by JNJ7777120. Moreover, H4R agonists induced an increase in phospho-Src levels detected by Western blot. Results revealed the involvement of phospho-Src in EMT events. Upon treatment with H4R agonists there was an increase in phospho-ERK1/2 and TGF-ß1 levels by Western blot, in Smad2/3 positive nuclei by indirect immunofluorescence, and in tumor spheres formation by the mammosphere assay. Notably, the selective TGF-ß1 kinase/activin receptor-like kinase inhibitor A83-01 blocked these effects. Moreover, cells derived from mammospheres exhibited higher Slug expression and enhanced migratory behavior. Collectively, findings support the interaction between H4R and TGF-ß receptor signaling in the enhancement of EMT features and mammosphere formation and point out intracellular TGF-ß1 as a potential mediator of these events.

The Yeast Hsp70 Cochaperone Ydj1 Regulates Functional Distinction of Ssa Hsp70s in the Hsp90 Chaperoning Pathway.

Heat-shock protein (Hsp) 90 assists in the folding of diverse sets of client proteins including kinases and growth hormone receptors. Hsp70 plays a major role in many Hsp90 functions by interacting and modulating conformation of its substrates before being transferred to Hsp90s for final maturation. Each eukaryote contains multiple members of the Hsp70 family. However, the role of different Hsp70 isoforms in Hsp90 chaperoning actions remains unknown. Using v-Src as an Hsp90 substrate, we examined the role of each of the four yeast cytosolic Ssa Hsp70s in regulating Hsp90 functions. We show that the strain expressing stress-inducible Ssa3 or Ssa4, and the not constitutively expressed Ssa1 or Ssa2, as the sole Ssa Hsp70 isoform reduces v-Src-mediated growth defects. The study shows that although different Hsp70 isoforms interact similarly with Hsp90s, v-Src maturation is less efficient in strains expressing Ssa4 as the sole Hsp70. We further show that the functional distinction between Ssa2 and Ssa4 is regulated by its C-terminal domain. Further studies reveal that Ydj1, which is known to assist substrate transfer to Hsp70s, interacts relatively weakly with Ssa4 compared with Ssa2, which could be the basis for poor maturation of the Hsp90 client in cells expressing stress-inducible Ssa4 as the sole Ssa Hsp70. The study thus reveals a novel role of Ydj1 in determining the functional distinction among Hsp70 isoforms with respect to the Hsp90 chaperoning action.

Up-regulated fibronectin in 3D culture facilitates spreading of triple negative breast cancer cells on 2D through integrin ß-5 and Src.

Using MDA-MB-231 cells as a model of triple negative breast cancer (TNBC) and its metastatic sub-cell lines that preferentially metastasize to lung, bone or brain, we found that the mRNA and protein levels of fibronectin (FN) are increased in MDA-MB-231 cells and its lung metastatic derivative, when cultivated in three-dimensional (3D) suspension cultures. The increase of FN expression in 3D was dependent on p38 mitogen-activated protein kinase (MAPK) because it was prevented by treatment of cells with SB203580, an inhibitor of p38MAPK. The up-regulated FN was converted into fibrils, and it enhanced cell spreading when cells cultured in 3D were transferred to two-dimensional (2D) culture. The arginine-glycine-aspartate (RGD) peptides and siRNAs targeting of integrin ß-5 inhibited spreading of cells regardless of the presence of FN on 2D culture dishes. In addition, the levels of phosphorylated Src were found to be increased in 3D and the treatment of cells with SU6656, an inhibitor of Src, decreased the rate of cell spreading on FN. Collectively, these studies demonstrate that increased cellular FN in 3D suspension culture facilitates cancer cell attachment and spreading via integrin ß-5 and Src, suggesting that the increased FN promotes initial attachment of cancer cells to secondary organs after circulation during metastasis.

Identification of an acetonitrile addition impurity formed during peptide disulfide bond reduction using dithiothreitol and Tris(2-carboxyethyl)phosphine.

Identification and localization of modifications in peptides containing multiple disulfide bonds is challenging due to inefficient fragmentation in mass spectrometry (MS) analysis. In cases where MS fragmentation techniques such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) fail to achieve efficient fragmentation, off-line disulfide bond reduction techniques are typically employed prior to MS analysis. Some commonly used reducing agents include dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In this work, we describe the detection and identification of an unexpected impurity that formed during the reduction of Peptide A, containing multiple disulfide bonds, while using DTT or TCEP as reducing agents and acetonitrile as a co-solvent. The DTT reduced products were found to be a mixture of the expected linear Peptide A (fully reduced) and an unknown product (>50%) with a mass corresponding to linear Peptide A plus 41 Da ([reduced-M + 41]). A series of experiments were subsequently performed to investigate the identity and origin of this impurity. Disulfide bond reduction with DTT was performed in aqueous mixtures containing acetonitrile, methanol, and deuterated acetonitrile; and with TCEP in aqueous mixtures containing acetonitrile. Additionally, glycine amino acid was used as a surrogate to investigate the mechanism. The liquid chromatography-mass spectrometry (LCMSMS) results demonstrated that the [reduced-M + 41] impurity was an acetonitrile addition on the peptide's N-terminal glycine. The corresponding impurity [M + 41] was also found in the native Peptide A (non-reduced), suggesting that small amounts of this impurity may also be generated during the synthesis in the upstream process steps. By understanding the formation of this process-related impurity [M + 41], one could potentially reduce or eliminate its presence in Peptide A through chemical controls. Finally, this observation provides caution against using acetonitrile as a co-solvent during DTT- or TCEP-promoted reduction of peptides with an uncapped N-terminus primary amine.

Synaptic Microtubule-Associated Protein EB3 and SRC Phosphorylation Mediate Structural and Behavioral Adaptations During Withdrawal From Cocaine Self-Administration.

Addictive behaviors, including relapse, are thought to depend in part on long-lasting drug-induced adaptations in dendritic spine signaling and morphology in the nucleus accumbens (NAc). While the influence of activity-dependent actin remodeling in these phenomena has been studied extensively, the role of microtubules and associated proteins remains poorly understood. We report that pharmacological inhibition of microtubule polymerization in the NAc inhibited locomotor sensitization to cocaine and contextual reward learning. We then investigated the roles of microtubule end-binding protein 3 (EB3) and SRC kinase in the neuronal and behavioral responses to volitionally administered cocaine. In synaptoneurosomal fractions from the NAc of self-administering male rats, the phosphorylation of SRC at an activating site was induced after 1 d of withdrawal, while EB3 levels were increased only after 30 d of withdrawal. Blocking SRC phosphorylation during early withdrawal by virally overexpressing SRCIN1, a negative regulator of SRC activity known to interact with EB3, abolished the incubation of cocaine craving in both male and female rats. Conversely, mimicking the EB3 increase observed after prolonged withdrawal increased the motivation to consume cocaine in male rats. In mice, the overexpression of either EB3 or SRCIN1 increased dendritic spine density and altered the spine morphology of NAc medium spiny neurons. Finally, a cocaine challenge after prolonged withdrawal recapitulated most of the synaptic protein expression profiles observed at early withdrawal. These findings suggest that microtubule-associated signaling proteins such as EB3 cooperate with actin remodeling pathways, notably SRC kinase activity, to establish and maintain long-lasting cellular and behavioral alterations following cocaine self-administration.SIGNIFICANCE STATEMENT Drug-induced morphological restructuring of dendritic spines of nucleus accumbens neurons is thought to be one of the cellular substrates of long-lasting drug-associated memories. The molecular basis of these persistent changes has remained incompletely understood. Here we implicate for the first time microtubule function in this process, together with key players such as microtubule-bound protein EB3 and synaptic SRC phosphorylation. We propose that microtubule and actin remodeling cooperate during withdrawal to maintain the plastic structural changes initially established by cocaine self-administration. This work opens new translational avenues for further characterization of microtubule-associated regulatory molecules as putative drug targets to tackle relapse to drug taking.

Accumulation of the myosin-II-spectrin complex plays a positive role in apical extrusion of Src-transformed epithelial cells.

At the initial stage of carcinogenesis, transformation occurs in single cells within the epithelium. Recent studies have revealed that the newly emerging transformed cells are often apically eliminated from epithelial tissues. However, the underlying molecular mechanisms of this cancer preventive phenomenon still remain elusive. In this study, we first demonstrate that myosin-II accumulates in Src-transformed cells when they are surrounded by normal epithelial cells. Knock-down of the heavy chains of myosin-II substantially diminishes apical extrusion of Src cells, suggesting that accumulated myosin-II positively regulates the apical elimination of transformed cells. Furthermore, we have identified ß-spectrin as a myosin-II-binding protein under the coculture of normal and Src-transformed epithelial cells. ß-spectrin is also accumulated in Src cells that are surrounded by normal cells, and the ß-spectrin accumulation is regulated by myosin-II. Moreover, knock-down of ß-spectrin significantly suppresses apical extrusion of Src cells. Collectively, these results indicate that accumulation of the myosin-II-spectrin complex plays a positive role in apical extrusion of Src-transformed epithelial cells. Further elucidation of the molecular mechanisms of apical extrusion would lead to the establishment of a novel type of cancer preventive medicine.

Quantitative biodistribution of biotherapeutics at whole body, organ and cellular levels by autoradiography.

AIM: Tools for mapping and quantifying monoclonal antibody (mAb) and peptide biotherapeutics distribumtion were evaluated by comparing data from three independent methods conducted at the whole body, organ or tissue, and cellular levels. MATERIALS & METHODS: [3H]-mAb1 and [3H]-peptide A were administered intravenously to rats followed by quantitative whole-body autoradiography, kidney macro-autoradiography and micro-autoradiography. RESULTS: [3H]-mAb1 and [3H]-peptide A concentrations were measured in anatomical regions ranging from whole body to whole organ to sub-organ level, such as the kidney glomerulus, with increasing resolution. The tissue/blood [3H]-mAb1 concentrations in selected kidney microenvironments were comparable among the three quantitative methods. CONCLUSION: Quantitative whole-body autoradiography, tissue macro-autoradiography and micro-autoradiography all provide useful tools for quantifying the concentrations of biotherapeutics at different anatomical levels in tissues, facilitating better predictions of efficacy and toxicity.

Chemical genetic inhibition of DEAD-box proteins using covalent complementarity.

DEAD-box proteins are an essential class of enzymes involved in all stages of RNA metabolism. The study of DEAD-box proteins is challenging in a native setting since they are structurally similar, often essential and display dosage sensitivity. Pharmacological inhibition would be an ideal tool to probe the function of these enzymes. In this work, we describe a chemical genetic strategy for the specific inactivation of individual DEAD-box proteins with small molecule inhibitors using covalent complementarity. We identify a residue of low conservation within the P-loop of the nucleotide-binding site of DEAD-box proteins and show that it can be mutated to cysteine without a substantial loss of enzyme function to generate electrophile-sensitive mutants. We then present a series of small molecules that rapidly and specifically bind and inhibit electrophile-sensitive DEAD-box proteins with high selectivity over the wild-type enzyme. Thus, this approach can be used to systematically generate small molecule-sensitive alleles of DEAD-box proteins, allowing for pharmacological inhibition and functional characterization of members of this enzyme family.

The tyrosine kinase v-Src causes mitotic slippage by phosphorylating an inhibitory tyrosine residue of Cdk1.

The nonreceptor tyrosine kinase v-Src is an oncogene first identified in Rous sarcoma virus. The oncogenic effects of v-Src have been intensively studied; however, its effects on chromosomal integrity are not fully understood. Here, using HeLa S3/v-Src cells having inducible v-Src expression, we found that v-Src causes mitotic slippage in addition to cytokinesis failure, even when the spindle assembly checkpoint is not satisfied because of the presence of microtubule-targeting agents. v-Src's effect on mitotic slippage was also observed in cells after a knockdown of C-terminal Src kinase (Csk), a protein-tyrosine kinase that inhibits Src-family kinases and was partially inhibited by PP2, an Src-family kinase inhibitor. Proteomic analysis and in vitro kinase assay revealed that v-Src phosphorylates cyclin-dependent kinase 1 (Cdk1) at Tyr-15. This phosphorylation attenuated Cdk1 kinase activity, resulting in a decrease in the phosphorylation of Cdk1 substrates. Furthermore, v-Src-induced mitotic slippage reduced the sensitivity of the cells to microtubule-targeting agents, and cells that survived the microtubule-targeting agents exhibited polyploidy. These results suggest that v-Src causes mitotic slippage by attenuating Cdk1 kinase activity via direct phosphorylation of Cdk1 at Tyr-15. On the basis of these findings, we propose a model for v-Src-induced oncogenesis, in which v-Src-promoted mitotic slippage due to Cdk1 phosphorylation generates genetic diversity via abnormal cell division of polyploid cells and also increases the tolerance of cancer cells to microtubule-targeting agents.

v-Src-driven transformation is due to chromosome abnormalities but not Src-mediated growth signaling.

v-Src is the first identified oncogene product and has a strong tyrosine kinase activity. Much of the literature indicates that v-Src expression induces anchorage-independent and infinite cell proliferation through continuous stimulation of growth signaling by v-Src activity. Although all of v-Src-expressing cells are supposed to form transformed colonies, low frequencies of v-Src-induced colony formation have been observed so far. Using cells that exhibit high expression efficiencies of inducible v-Src, we show that v-Src expression causes cell-cycle arrest through p21 up-regulation despite ERK activation. v-Src expression also induces chromosome abnormalities and unexpected suppression of v-Src expression, leading to p21 down-regulation and ERK inactivation. Importantly, among v-Src-suppressed cells, only a limited number of cells gain the ability to re-proliferate and form transformed colonies. Our findings provide the first evidence that v-Src-driven transformation is attributed to chromosome abnormalities, but not continuous stimulation of growth signaling, possibly through stochastic genetic alterations.

Syntenin mediates SRC function in exosomal cell-to-cell communication.

The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin-syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell-cell communication, supported by syntenin exosomes, which is likely to contribute to tumor-host interactions.