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David Cowburn – One of the best experts on this subject based on the ideXlab platform.
Abl Kinase constructs expressed in bacteria: facilitation of structural and functional studies including segmental labeling by expressed protein ligationMolecular bioSystems, 2012Co-Authors: Dongsheng Liu, David CowburnAbstract:
A great portion of tyrosine Kinases are involved in cell development and their structural alteration is intimately involved in associated pathologies of development and oncology. These Kinases are one of the major groups of targets under investigation for molecular therapeutics. To carry out biochemical and structural biological studies on these Kinases, economical production of their purified forms is highly desirable. However over-expressing tyrosine Kinases as recombinant forms in bacterial systems and their purification is a significant challenge. Abelson Kinase (Abl) has previously been expressed on a large scale to facilitate X-ray crystallography and NMR structure studies mainly in baculovirus infected insect cells. Even though success has been achieved in expression of soluble tyrosine Kinases in E. coli with chaperones to improve correct folding, low expression levels of Kinases are intrinsic in such systems because of diversion of resources to produce chaperones. Here we present a straightforward method to express and purify isolated Abl Kinase domain and SH3–SH2–Kinase multi-domain structures. The expressed Abl protein retains its correct folding and biological function. The yield of soluble protein is in a several mg L−1 range in minimal media. Furthermore we demonstrate that segmental isotopic labelling using expressed protein ligation can be achieved using bacterial expressed Abl Kinase domain constructs, which is especially useful in NMR structure–activity studies.
determination of the rotational diffusion tensor of macromolecules in solution from nmr relaxation data with a combination of exact and approximate methods application to the determination of interdomain orientation in multidomain proteinsJournal of Magnetic Resonance, 2001Co-Authors: Ranajeet Ghose, David Fushman, David CowburnAbstract:
In this paper we present a method for determining the rotational diffusion tensor from NMR relaxation data using a combination of approximate and exact methods. The approximate method, which is computationally less intensive, computes values of the principal components of the diffusion tensor and estimates the Euler angles, which relate the principal axis frame of the diffusion tensor to the molecular frame. The approximate values of the principal components are then used as starting points for an exact calculation by a downhill simplex search for the principal components of the tensor over a grid of the space of Euler angles relating the diffusion tensor frame to the molecular frame. The search space of Euler angles is restricted using the tensor orientations calculated using the approximate method. The utility of this approach is demonstrated using both simulated and experimental relaxation data. A quality factor that determines the extent of the agreement between the measured and predicted relaxation data is provided. This approach is then used to estimate the relative orientation of SH3 and SH2 domains in the SH(32) dual-domain construct of Abelson Kinase complexed with a consolidated ligand.
Solid-Phase Synthesis of Consolidated Ligands Containing an Intramolecular Lactam Bridge: Comparison of Strategies and TacticsPeptides: The Wave of the Future, 2001Co-Authors: Jaya T. Varkey, David Cowburn, George BaranyAbstract:
Src homology (SH) domains are building blocks involved in intracellular signal transduction . Previous work has defined and demonstrated consolidated ligands, which combine in the same molecule peptide sequences recognized by SH2 and SH3 domains (i.e., Pro-Val-pTyr-Glu-Asn-Val and Pro-Pro-Ala-Tyr-Pro-Pro-Pro-Pro-Val-Pro, respectively), and exhibit enhanced affinities and specificities towards dual SH(32) Abelson Kinase [2,3]. For first-generation consolidated ligands, binding sequences were connected by a flexible linker, e.g., Glyn. With the goal to further improve their efficacies, several second-generation consolidated ligands with a more rigid linker, e.g., Alan, and optionally including an intramolecular lactam bridge “lock”, were designed and synthesized . The present report focuses on the 32-residue structure shown below, which contains an i to i+7 lactam bridge to connect the side-chains of Glu and Lys, with an intervening 5-aminovaleric acid (Ava) as a spacer. Two different solid-phase synthetic strategies were evaluated.
Mark Peifer – One of the best experts on this subject based on the ideXlab platform.
The Crk adapter protein is essential for Drosophila embryogenesis, where it regulates multiple actin-dependent morphogenic events.Molecular biology of the cell, 2019Co-Authors: Andrew J. Spracklen, Emma M. Thornton-kolbe, Alison N. Bonner, Alexandru Florea, Peter J. Compton, Rodrigo Fernandez-gonzalez, Mark PeiferAbstract:
Small Src homology domain 2 (SH2) and 3 (SH3) adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The CT10 regulator of Kinase (Crk) family has tissue-specific roles in phagocytosis, cell migration, and neuronal development and mediates oncogenic signaling in pathways like that of Abelson Kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk’s full role in embryogenesis. We circumvented these limitations with short hairpin RNA and CRISPR technology to assess Crk’s function in Drosophila morphogenesis. We found that Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates cortical localization of the actin-related protein 2/3 complex (Arp2/3), its regulator suppressor of cAMP receptor (SCAR), and filamentous actin to actin caps and pseudocleavage furrows. Crk loss leads to the loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.
The Crk adapter protein is essential for Drosophila embryogenesis, where it regulates multiple actin-dependent morphogenic events, 2019Co-Authors: Andrew J. Spracklen, Emma M. Thornton-kolbe, Alison N. Bonner, Alexandru Florea, Peter J. Compton, Rodrigo Fernandez-gonzalez, Mark PeiferAbstract:
Abstract Small SH2/SH3 adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The Crk family has tissue-specific roles in phagocytosis, cell migration and neuronal development, and mediates oncogenic signaling in pathways like that of Abelson Kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk’s full role in embryogenesis. We circumvented these limitations with shRNA and CRISPR technology to assess Crk’s function in Drosophila morphogenesis. We found Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates levels of the Arp2/3 complex, its regulator SCAR, and F-actin in actin caps and pseudocleavage furrows. Crk loss leads to loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics.
Abelson Kinase Acts as a Robust, Multifunctional Scaffold in Regulating Embryonic MorphogenesisMolecular biology of the cell, 2016Co-Authors: Edward M. Rogers, Andrew J. Spracklen, Colleen G. Bilancia, Kaelyn D. Sumigray, S. Colby Allred, Stephanie H. Nowotarski, Kristina N. Schaefer, Benjamin J. Ritchie, Mark PeiferAbstract:
Abelson family Kinases (Abls) are key regulators of cell behavior and the cytoskeleton during development and in leukemia. Abl’s SH3, SH2, and tyrosine Kinase domains are joined via a linker to an F-actin-binding domain (FABD). Research on Abl’s roles in cell culture led to several hypotheses for its mechanism of action: 1) Abl phosphorylates other proteins, modulating their activity, 2) Abl directly regulates the cytoskeleton via its cytoskeletal interaction domains, and/or 3) Abl is a scaffold for a signaling complex. The importance of these roles during normal development remains untested. We tested these mechanistic hypotheses during Drosophila morphogenesis using a series of mutants to examine Abl’s many cell biological roles. Strikingly, Abl lacking the FABD fully rescued morphogenesis, cell shape change, actin regulation, and viability, whereas Kinase-dead Abl, although reduced in function, retained substantial rescuing ability in some but not all Abl functions. We also tested the function of four conserved motifs in the linker region, revealing a key role for a conserved PXXP motif known to bind Crk and Abi. We propose that Abl acts as a robust multidomain scaffold with different protein motifs and activities contributing differentially to diverse cellular behaviors.
Naoki Watanabe – One of the best experts on this subject based on the ideXlab platform.
Abi-1-bridged tyrosine phosphorylation of VASP by Abelson Kinase impairs association of VASP to focal adhesions and regulates leukaemic cell adhesion.The Biochemical journal, 2012Co-Authors: Masahiro Maruoka, Yunfeng Yuan, Mizuho Sato, Tatsuo Takeya, Takuya Ogawa, Norihiro Ishida-kitagawa, Masayoshi Ichiba, Ryosuke Fujii, Naoki WatanabeAbstract:
Mena [mammalian Ena (Enabled)]/VASP (vasodilator-stimulated phosphoprotein) proteins are the homologues of Drosophila Ena. In Drosophila, Ena is a substrate of the tyrosine Kinase DAbl (Drosophila Abl). However, the link between Abl and the Mena/VASP family is not fully understood in mammals. We previously reported that Abi-1 (Abl interactor 1) promotes phosphorylation of Mena and BCAP (B-cell adaptor for phosphoinositide 3-Kinase) by bridging the interaction between c-Abl and the substrate. In the present study we have identified VASP, another member of the Mena/VASP family, as an Abi-1-bridged substrate of Abl. VASP is phosphorylated by Abl when Abi-1 is co-expressed. We also found that VASP interacted with Abi-1 both in vitro and in vivo. VASP was tyrosine-phosphorylated in Bcr-Abl-positive leukaemic cells in an Abi-1-dependent manner. Co-expression of c-Abl and Abi-1 or the phosphomimetic Y39D mutation in VASP resulted in less accumulation of VASP at focal adhesions. VASP Y39D had a reduced affinity to the proline-rich region of zyxin. Interestingly, overexpression of both phosphomimetic and unphosphorylated forms of VASP, but not wild-type VASP, impaired adhesion of K562 cells to fibronectin. These results suggest that the phosphorylation and dephosphorylation cycle of VASP by the Abi-1-bridged mechanism regulates association of VASP with focal adhesions, which may regulate adhesion of Bcr-Abl-transformed leukaemic cells.
Imatinib mesylate (STI571)-induced cell edge translocation of Kinase-active and Kinase-defective Abelson Kinase: requirements of myristoylation and src homology 3 domain.Molecular pharmacology, 2008Co-Authors: Akiko Fujita, Tomoyuki Shishido, Yunfeng Yuan, Eiji Inamoto, Shuh Narumiya, Naoki WatanabeAbstract:
4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate (STI571) is the first successful target-based drug with excellent potency against chronic myelogenous leukemia. Studies on this compound have illuminated potentials and problems of Kinase inhibitors in the treatment of cancer. As found in crystal structures, STI571-bound Abelson Kinase (abl) is believed to form closed conformation with N-terminal regulatory domains. Here we present evidence of distinct STI571-induced modulation of abl functions using high-resolution live-cell imaging approaches. Within lamellipodia of fibroblast cells, STI571 was found to induce rapid translocation of abl to the lamellipodium tip. Quantitative analysis yielded 0.81 and 1.8 μM for EC50 values of STI571-induced cell edge translocation of abl-KD-green fluorescent protein (GFP) and wild-type abl-GFP, respectively. It also revealed adverse response of drug-resistant abl-T334I to STI571, suggesting that drug binding to abl-GFP triggers translocation. N-myristoylation and the src homology 3 (SH3) domain were required for this translocation, whereas disruption of intramolecular interactions of these motifs enhanced cell-edge association of abl. An intact C-terminal last exon region in abl, but not its F-actin binding, was required for efficient cell-edge translocation. Moreover, single-molecule observation revealed an STI571-induced rapid increase in slow diffusive species of abl in both the tip and the body region of lamellipodia. These results suggest that although activated abl translocates to the cell edge at its open state, STI571 can also bind and lock abl in the open and membrane-tethered conformation as long as the SH3 domain and the C-terminal region are intact. High-resolution imaging can be a powerful tool for elucidating inhibitor modulation of abl functions under intracellular environment.