No description available.

Thesis (Sc.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The c-Jun NH2-terminal kinase (JNK) and p38 intracellular signaling cascades are mitogen-activated protein kinase (MAPK) signaling pathways that are activated in mammalian cells by a variety of stressors, including proinflammatory cytokines, osmotic shock, and shear stress. The purpose of this dissertation research was to examine the effect of injury-producing exercise on JNK and p38 activities in human skeletal muscle and to determine whether mechanical stress is a primary stimulator of JNK and p38 activities with contraction. Twelve healthy subjects (7M/5F) completed maximal concentric or eccentric knee extensions on an isokinetic dynamometer (10 sets, 10 reps). Needle biopsies were obtained from the vastus lateralis muscle 24 h before exercise, immediately post-exercise, and 6 h post-exercise. While both forms of exercise increased JNK activity immediately post-exercise, eccentric contractions resulted in a much higher activation (15-fold vs. 4-fold increase above basal for eccentric and concentric, respectively). By 6 h post-exercise, JNK activity decreased back to baseline values. In a separate study, 14 male subjects completed a 42.2 km marathon. Biopsies were obtained from the vastus lateralis muscle 10 days prior to the marathon, immediately following the race, and 1, 3, and 5 days after the race. JNK activity increased 7-fold over basal immediately postexercise, but decreased back to basal 1, 3, and 5 days after the exercise. The activity of p38y also was increased and decreased in a similar pattern. However, no regulation was observed for p38α. In a third study, the effects of contraction and static stretch on JNK activity and p38 phosphorylation were determined in the rat soleus muscle in vitro. Static stretch dramatically increased JNK activity and p38 phosphorylation, whereas isometric contraction resulted in much smaller increases in JNK activity and p38 phosphorylation. The regulation of focal adhesion proteins also was examined following both exercise and contraction. The work presented in this thesis demonstrates that injury-producing exercise results in the marked activation of the JNK and p38 stress-activated protein kinases and provides evidence that mechanical stress may be a major contributor to increases in JNK and p38 activities observed following contraction in rat and human skeletal muscle. / 2031-01-01

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. October 2014. / The c-Jun N-terminal kinases (JNKs) are mitogen-activated protein kinases (MAPKs) that are activated by the dual phosphorylation of a canonical threonine and tyrosine residue. While it is well known that the activation of JNK mediates many important cellular processes such as differentiation, proliferation, and apoptosis, the mechanisms by which phosphorylation induces its activation are not known. An understanding of the structural and biophysical basis for the activation of JNK is highly desirable however, as dysregulation of the kinase has been implicated in numerous prominent diseases. Aiming first to improve upon the previously reported inadequacies in acquiring active JNK, this work describes a novel method for the purification of large yields of pure and phosphorylated JNK1β1, the most abundant JNK isoform. Using codon harmonization as a precautionary measure toward increasing the soluble overexpression of the kinase raised unique questions about the role of translation kinetics in both the heterologous and natural co-translational modification of kinases. After purifying the upstream activating kinases of JNK, phosphorylation of JNK1β1 was achieved by reconstituting the MEKK1 → MKK4 → JNK MAPK activation cascade in vitro. Activated JNK1β1 was thereafter able to phosphorylate its substrate, ATF2, with high catalytic efficiency. Characterising the nature of JNK1β1 modification by MKK4, mass spectrometry revealed that the latter kinase phosphorylates JNK1β1 not only at its activation residues (T183 and Y185), but also at a recognised yet uncharacterised phospho-site (S377) as well as two novel phospho-residues (T228 and S284) whose phosphorylation appear to have functional significance. Unfolding studies and amide hydrogen-deuterium exchange (HX) mass spectrometry (MS) were then used to investigate the changes to the stability and structure/conformational dynamics of JNK1β1 induced by phosphorylation and nucleotide substrate binding. Increased flexibility detected at the hinge between the N- and C-terminal domains upon phosphorylation suggested that activation may require interdomain closure. Patterns of solvent protection by the ATP analogue, AMP-PNP, reflected a novel mode of nucleotide binding to the C-terminal domain of a destabilised and open domain conformation of inactive JNK1β1. HX protection at both domains following AMP-PNP binding to active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, simultaneously stabilising the kinase. This reveals that phosphorylation activates JNK1β1 in part by enhancing the flexibility of the hinge to enable interdomain closure and the formation of a functional active site. This work thus offers novel insight into the unique molecular mechanisms by which JNK1β1 is regulated by nucleotide binding and phosphorylation by MKK4, and by the complex interplay that exists between them.

Accurate chromosome segregation requires a spindle apparatus composed of microtubules that arise from the spindle to attach to the kinetochore, a protein complex assembled at the centromere of each chromosome. Failure to segregate chromosomes accurately may lead to lethal early developmental defects and tumorigenesis. To achieve proper kinetochore binding to microtubules, mammalian cells have evolved elaborate mechanisms to correct attachment errors and stabilize correct ones. Current models suggest that tension between kinetochore pairs (inter-kinetochore stretch) and tension at the kinetochore (intra-kinetochore stretch) produces a spatial separation of Aurora B kinase from kinetochore-associated and microtubule-binding substrates, subsequently reducing their phosphorylations and increasing their microtubule affinity. However, the tension-based models do not explain how the initial microtubule binding at unattached kinetochores occurs, where there is no tension and kinetochore-associated substrates are highly phosphorylated and, hence unable to bind to microtubules. Therefore, there must be a mechanism that explains how the phosphorylation of kinetochore substrates by Aurora B is reduced in the absence of tension. In the first part of this thesis, I examine the structural features of the coiled-coil domain of the kinetochore-associated kinesin motor protein, CENP-E. Using Single-Molecule High-Resolution Colocalization (SHREC) microscopy analysis of kinetochore-associated CENP-E, I show that CENP-E undergoes structural rearrangements prior to and after tension generation at the kinetochore. Chemical inhibition of the motor motility or genetic perturbations of the coiled-coil domain of CENP-E increases Aurora B-mediated Ndc80 phosphorylation in a tension-independent manner. Importantly, metaphase chromosome misalignment caused by inhibition of CENP-E can be rescued by chemical inhibition of Aurora B kinase. Therefore, CENP-E regulates the initial kinetochore binding to microtubules and the stabilization of kinetochore-microtubule attachments. Formin-dependent actin assembly is known to play a role in multiple processes, including cytokinesis, filopodia formation, cell polarity, and cell adhesion. Thus, formin malfunction is directly linked to various pathologies, including defects in cell migration and tumor suppression. Although the role of formins in actin polymerization has been well described, the mechanistic processes that regulate the actin assembly function of formins remain poorly understood, especially the interplay among the various sub-families of formins and how they are spatiotemporally regulated. In the second part of this thesis, I show that Aurora B-mediated phosphorylation of the formin, mDia3 regulates actin assembly. Previous studies identified two Aurora B phosphorylation sites in the FH2 domain of mDia3. To this end, phosphomimetic and non-phosphorylatable mutants of a constitutively active form of mDia3 were designed to test whether phosphorylation by Aurora B regulates actin assembly. Using an in vitro actin polymerization kinetic assay and expression of fluorescently-tagged constitutively active mDia3 in cells, I show that phosphorylation of mDia3 by Aurora B induces the actin assembly function of mDia3. Furthermore, using a phospho-specific antibody, I show that mDia3 is phosphorylated by Aurora B. Live-cell analysis shows that perturbations of these phosphorylation sites affect cell migration and cell spreading. Therefore, I illustrate a novel regulatory mechanism for the actin assembly function of mDia3 that is dependent on Aurora B kinase activity.

M.Sc. (Biochemistry) / Please refer to full text to view abstract

No description available.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 64-72).

Thesis (M.S. in Bio.)--School of Biology, Georgia Institute of Technology, 2004. Directed by Harish Radhakrishna. / Includes bibliographical references (leaves 55-65).

Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 84-91). Also available for download via the World Wide Web; free to University of Oregon users.

The c-Jun N-terminal kinases (JNKs) are MAPK family members and are activated by stress, growth factors and cytokines. They are encoded by three separate genes (jnk 1, 2, and 3), spliced alternately creating 10 isoforms. JNK signaling promotes both cell death and cell survival in a stimuli and tissue specic manner and is also implicated in tumorigenesis. Using the Polyoma Virus Middle T Antigen (PyVMT) transgenic mouse model where jnk2 was either expressed or deleted, we found that the PyVMTjnk2-/- tumors expressed higher Epidermal Growth Factor Receptor Substrate 8 (EPS8) mRNA and protein. EPS8 regulates EGFR signaling from Ras to Rac and EGFR tracking via Rab5 and RN-Tre. EPS8 is a prime candidate for connecting the EGFR signaling to actin cytoskeleton remodeling, thus mediating cell migration, a critical step in metastasis. In migration assays, PyVMTjnk2+/+ cells migrated ve fold more than the PyVMTjnk2-/- cells. Re-expression of JNK2[alpha] in the PyVMTjnk2-/- cells rescued this phenotype. Expression of shRNA EPS8 in the PyVMTjnk2-/- cell increased migration in vitro. EPS8 localization at dorsal rues and internalization of EGF-EGFR complexes coincided with JNK2 expression. Expression of shEPS8 in the PyVMTjnk2-/- cells increased EGF internalization suggesting that in absence of JNK2, EPS8 participates in Rab5-RN-Tre complex that inhibits EGFR internalization. Finally, we report that in absence of JNK2, EPS8 protein stability is greatly increased, suggesting that JNK2 is essential for endosomal sorting and degradation of EGFR associated cargo, of which EPS8 is a critical part. In contrast, silencing JNK1 (p46) in 4T1.2 mammary tumor cells, consistently enhanced cell invasion and tumor growth. Tumors derived from orthotopic injection of the 4T1.2shJNK1 expressing cells into the mammary fat pad reached target volume signicantly earlier than non-silencing vector expressing tumors. When injected intravenously, signicantly higher lung metastasis was observed in the 4T1.2shJNK1 group. The more aggressive behavior of 4T1.2shJNK1 tumors was associated with an increase in CCR5 and pAkt as detected by microarray analysis. Taken together, our data suggest that JNK1 suppresses the expression of proteins associated with tumor growth and invasive phenotype, contributing to tumor progression. / text