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Kim K.,Incheon National University | Yang D.K.,Mount Sinai School of Medicine | Kim S.,New Drug Development Research Center Inc. | Kang H.,Incheon National University
Journal of Cellular Biochemistry | Year: 2015

The transforming growth factor β (TGFβ) signaling pathway is critical for the promotion and maintenance of the contractile phenotype of vascular smooth muscle cells (VSMCs). Though multiple microRNAs (miRNAs) implicated in the regulation of the VSMC phenotype have been identified, the modulation of miRNAs in the VSMCs by TGFβ signaling has not been fully described. In this study, we identified microRNA-142-3p (miR-142-3p) as a modulator of the VSMC phenotype in response to TGFβ signaling. We show that miR-142-3p is induced upon TGFβ signaling, leading to the repression of a novel target, dedicator of cytokinesis 6 (DOCK6). The downregulation of DOCK6 by miR-142-3p is critical for cell migration. Thus, this study demonstrates that miR-142-3p is a key regulator of the TGFβ-mediated contractile phenotype of VSMCs that acts through inhibiting cell migration through targeting DOCK6. J. Cell. Biochem. 116: 2325-2333, 2015. © 2015 Wiley Periodicals, Inc.

Zorba A.,Howard Hughes Medical Institute | Buosi V.,Howard Hughes Medical Institute | Kutter S.,Howard Hughes Medical Institute | Kern N.,Howard Hughes Medical Institute | And 4 more authors.
eLife | Year: 2014

We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX21-25. While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases. © Booth et al.

Cho H.J.,Yonsei University | Gee H.Y.,Yonsei University | Baek K.-H.,Yonsei University | Ko S.-K.,Yonsei University | And 5 more authors.
Journal of the American Chemical Society | Year: 2011

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cell-surface anion channel that permeates chloride and bicarbonate ions. The most frequent mutation of CFTR that causes cystic fibrosis is the deletion of phenylalanine at position 508 (ΔF508), which leads to defects in protein folding and cellular trafficking to the plasma membrane. The lack of the cell-surface CFTR results in a reduction in the lifespan due to chronic lung infection with progressive deterioration of lung function. Hsc70 plays a crucial role in degradation of mutant CFTR by the ubiquitin-proteasome system. To date, various Hsc70 inhibitors and transcription regulators have been tested to determine whether they correct the defective activity of mutant CFTR. However, they exhibited limited or questionable effects on restoring the chloride channel activity in cystic fibrosis cells. Herein, we show that a small molecule apoptozole (Az) has high cellular potency to promote membrane trafficking of mutant CFTR and its chloride channel activity in cystic fibrosis cells. Results from affinity chromatography and ATPase activity assay indicate that Az inhibits the ATPase activity of Hsc70 by binding to its ATPase domain. In addition, a ligand-directed protein labeling and molecular modeling studies also suggest the binding of Az to an ATPase domain, in particular, an ATP-binding pocket. It is proposed that Az suppresses ubiquitination of ΔF508-CFTR maybe by blocking interaction of the mutant with Hsc70 and CHIP, and, as a consequence, it enhances membrane trafficking of the mutant. © 2011 American Chemical Society.

Pai J.,Yonsei University | Yoon T.,Yonsei University | Kim N.D.,New Drug Development Research Center Inc. | Lee I.-S.,Konkuk University | And 2 more authors.
Journal of the American Chemical Society | Year: 2012

A rapid and quantitative method to evaluate binding properties of hairpin RNAs to peptides using peptide microarrays has been developed. The microarray technology was shown to be a powerful tool for high-throughput analysis of RNA-peptide interactions by its application to profiling interactions between 111 peptides and six hairpin RNAs. The peptide microarrays were also employed to measure hundreds of dissociation constants (Kd) of RNA-peptide complexes. Our results reveal that both hydrophobic and hydrophilic faces of amphiphilic peptides are likely involved in interactions with RNAs. Furthermore, these results also show that most of the tested peptides bind hairpin RNAs with submicromolar Kd values. One of the peptides identified by using this method was found to have good inhibitory activity against TAR-Tat interactions in cells. Because of their great applicability to evaluation of nearly all types of RNA-peptide interactions, peptide microarrays are expected to serve as robust tools for rapid assessment of peptide-RNA interactions and development of peptide ligands against RNA targets. © 2012 American Chemical Society.

Choi W.-S.,Rockefeller University | Choi W.-S.,New Drug Development Research Center Inc. | Rice W.J.,New York Structural Biology Center | Stokes D.L.,New York Structural Biology Center | And 2 more authors.
Blood | Year: 2013

ntegrin αIIbβ3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified αIIbβ3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away fromthemembrane.Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the aIIb lower leg is bent between the calf-1 and calf-2 domains and the β3 Integrin-Epidermal Growth Factor (I-EGF) 2 to 4 domains are freely coiled rather than in a cleft between the β3 headpiece and the aIIb lower leg. Our data indicate an important role for the region that links the distal calf-2 and b-tail domains to their respective transmembrane (TM) domains in transmitting the conformational changes in the TM domains associated with inside-out activation. © 2013 by The American Society of Hematology.

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