Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation.

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Electron-beam lithography and molecular liftoff for directed attachment of DNA nanostructures on silicon: top-down meets bottom-up.

CONSPECTUS: Our work on lithographic patterning of DNA nanostructures was inspired by a collaboration on molecular electronic devices known as quantum-dot cellular automata or QCA. QCA is a paradigm for computation in which information is transmitted and processed through the interaction of coupled electrical charges or magnetic dipoles. We began to explore the idea of molecular scale QCA and found that ab initio methods, a thermodynamic Ising model, and larger scale circuit design work suggested that circuits that did computationally interesting things could function at room temperature if made from molecular QCA cells of chemically reasonable design. But how could the QCA cells be patterned to form the complex arrays needed for computationally interesting circuitry, and how could those arrays of molecular circuitry be integrated with conventional electronic inputs and outputs? Top-down methods lacked the spatial resolution and high level of parallelism needed to make molecular circuits. Bottom-up chemical synthesis lacked the ability to fabricate arbitrary and heterogeneous structures tens to hundreds of nanometers in size. Chemical self-assembly at the time could produce structures in the right size scale, but was limited to homogeneous arrays. A potential solution to this conundrum was just being demonstrated in the late 1990s and early 2000s: DNA nanostructures self-assembled from oligonucleotides, whose high information density could handle the creation of arbitrary structures and chemical inhomogeneity. Our group became interested in whether DNA nanostructures could function as self-assembling circuit boards for electrical or magnetic QCA systems. This Account focuses on what we learned about the interactions of DNA nanostructures with silicon substrates and, particularly, on how electron-beam lithography could be used to direct the binding of DNA nanostructures on a variety of functional substrates.

Induction of myeloproliferative disorder and myelofibrosis by thrombopoietin receptor W515 mutants is mediated by cytosolic tyrosine 112 of the receptor.

Constitutively active JAK2V617F and thrombopoietin receptor (TpoR) W515L/K mutants are major determinants of human myeloproliferative neoplasms (MPNs). We show that a TpoRW515 mutation (W515A), which we detected in 2 myelofibrosis patients, and the Delta5TpoR active mutant, where the juxtamembrane R/KW(515)QFP motif is deleted, induce a myeloproliferative phenotype in mouse bone marrow reconstitution experiments. This phenotype required cytosolic Y112 of the TpoR. Phosphotyrosine immunoprofiling detected phosphorylated cytosolic TpoR Y78 and Y112 in cells expressing TpoRW515A. Mutation of cytosolic Y112 to phenylalanine prevented establishment of the in vivo phenotype and decreased constitutive active signaling by Delta5TpoR and TpoRW515A, especially via the mitogen-activated protein (MAP)-kinase pathway, without decreasing Janus kinase 2 (JAK2) activation. In contrast, mutation of cytosolic Y78 to phenylalanine enhanced the myeloproliferative syndrome induced by the TpoRW515 mutants, by enhancing receptor-induced JAK2 activation. We propose that TpoR cytosolic phosphorylated Y112 and flanking sequences could become targets for pharmacologic inhibition in MPNs.

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL.

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

Structure determination and infrared spectroscopy of K(UO2)(SO4)(OH)(H2O) and K(UO2)(SO4)(OH).

Two potassium uranyl sulfate compounds were synthesized, and their crystal structures were determined by single-crystal X-ray diffraction. K(UO2)(SO4)(OH)(H2O) (KUS1) crystallizes in space group P21/c, a = 8.0521(4) A, b = 7.9354(4) A, c = 11.3177(6) A, beta = 107.6780(10) degrees , V = 689.01(6) A3, and Z = 4. K(UO2)(SO4)(OH) (KUS2) is orthorhombic Pbca, a = 8.4451(2) A, b = 10.8058(4) A, c = 13.5406(5)A, V = 1235.66(7)A3, and Z = 8. Both structures were refined on the basis of F2 for all unique data collected with Mo Kalpha radiation and a CCD-based detector to agreement indices R1 = 0.0251 and 0.0206 calculated for 2856 and 2616 reflections for KUS1 and KUS2, respectively. The structures contain vertex-sharing uranyl pentagonal bipyramids and sulfate tetrahedra linked into new chains and sheet topologies. Infrared spectroscopy provides additional information about the linkages between the sulfate and uranyl polyhedra, as well as the hydrogen bonding present in the structures. The U-O-S connectivity is examined in detail, and the local bond angle is impacted by the steric constraints of the crystal structure.

A novel fusion of RBM6 to CSF1R in acute megakaryoblastic leukemia.

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.

Phosphotyrosine profiling identifies the KG-1 cell line as a model for the study of FGFR1 fusions in acute myeloid leukemia.

The 8p11 myeloproliferative syndrome (EMS) is associated with translocations that disrupt the FGFR1 gene. To date, 8 fusion partners of FGFR1 have been identified. However, no primary leukemia cell lines were identified that contain any of these fusions. Here, we screened more than 40 acute myeloid leukemia cell lines for constitutive phosphorylation of STAT5 and applied an immunoaffinity profiling strategy to identify tyrosine-phosphorylated proteins in the KG-1 cell line. Mass spectrometry analysis of KG-1 cells revealed aberrant tyrosine phosphorylation of FGFR1. Subsequent analysis led to the identification of a fusion of the FGFR1OP2 gene to the FGFR1 gene. Small interfering RNA (siRNA) against FGFR1 specifically inhibited the growth and induced apoptosis of KG-1 cells. Thus, the KG-1 cell line provides an in vitro model for the study of FGFR1 fusions associated with leukemia and for the analysis of small molecule inhibitors against FGFR1 fusions.

Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib.

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.

Activating alleles of JAK3 in acute megakaryoblastic leukemia.

Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.

A common phosphotyrosine signature for the Bcr-Abl kinase.

The Bcr-Abl fusion kinase drives oncogenesis in chronic myeloid leukemia (CML). CML patients are currently treated with the Abl tyrosine kinase inhibitor imatinib, which is effective in early stages of the disease. However, resistance to imatinib arises in later disease stages primarily because of a Bcr-Abl mutation. To gain deeper insight into Bcr-Abl signaling pathways, we generated phosphotyrosine profiles for 6 cell lines that represent 3 Bcr-Abl fusion types by using immunoaffinity purification of tyrosine phosphopeptides followed by tandem mass spectrometry. We identified 188 nonredundant tyrosine-phosphorylated sites, 77 of which are novel. By comparing the profiles, we found a number of phosphotyrosine sites common to the 6 cell lines regardless of cellular background and fusion type, several of which are decreased by imatinib treatment. Comparison of this Bcr-Abl signature with the profile of cells expressing an alternative imatinib-sensitive fusion kinase, FIP1L1-PDGFRalpha, revealed that these kinases signal through different pathways. This phosphoproteomic study of the Bcr-Abl fusion kinase highlights novel disease markers and potential drug-responsive biomarkers and adds novel insight into the oncogenic signals driven by the Bcr-Abl kinase.

Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes.

STAT5 is constitutively phosphorylated in leukemic cells in approximately 70% of acute myeloid leukemia (AML) patients. To identify kinase candidates potentially responsible for STAT5 phosphorylation, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) mass spectrometry to detect phosphoproteins in AML cell lines. We established TEL-ARG and BCR-ABL fusion proteins as the mechanism underlying STAT5 phosphorylation in HT-93 and KBM-3 cells, respectively. In addition, we identified a JAK2 pseudokinase domain mutation in HEL cells and using siRNA downregulation, established JAK2 as the kinase responsible for phosphorylating STAT5. This study illustrates the benefit of LC-MS/MS mass spectrometry and siRNA for the identification of novel targets and mutations.

Evaluation of CML model cell lines and imatinib mesylate response: determinants of signaling profiles.

Our understanding of the mechanisms by which BCR-ABL drives CML is based, in part, on the use of model cell lines such as the K562 cell line. However, the BCR-ABL translocation may occur via a number of different junction points. In addition, CML is a disease of hematopoietic stem cells and, as a result, can give rise to multiple lineages of tumor cells. In this study, we examined the cellular signaling profiles following imatinib mesylate treatment of eight model CML and ALL cell lines that encompass three BCR-ABL junction points and multiple lineages. We used phosphorylation-specific antibodies and flow cytometry to determine the kinase and pathway activation states with each of the cell lines before and after imatinib mesylate exposure. The comparisons of signaling response profiles, junction points and lineages indicate that cell line lineage rather than BCR-ABL junction point may determine cellular response to imatinib mesylate. The large amount of variation observed among the cell lines suggests that further analysis is required to understand the complex signaling profiles present in CML patients.

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway.

Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.

SAPK/JNK regulates cdc2/cyclin B kinase through phosphorylation and inhibition of cdc25c.

Cells undergo M phase arrest in response to stresses like UV irradiation or DNA damage. Stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase, JNK) is activated by such stress stimuli. We addressed the potential effects of SAPK activation on cell cycle regulatory proteins. Activation of SAPK strongly correlated with inhibition of cdc2/cyclin B kinase, an important regulator of G2/M phase. SAPK directly phosphorylated the cdc2 regulator, cdc25c, in vitro on serine 168 (S168). This residue was highly phosphorylated in vivo in response to stress stimuli. cdc25c phosphorylated on S168 in cells lacks phosphatase activity, and expression of a S168A mutant of cdc25c reversed the inhibition of cdc2/cyclin B kinase activity by cell stress. Antibodies directed against phosphorylated S168 detect increased phosphorylation of S168 after cell stress. We conclude that SAPK regulates cdc2/cyclin B kinase following stress events by a novel mechanism involving inhibitory phosphorylation of the cdc2-activating phosphatase cdc25c on S168.