The reversible phosphorylation of proteins plays a key role in nearly every aspect of cell life. This essential post-translational modification controls a myriad of cellular events from cell survival, differentiation, and migration to apoptosis. Two classes of enzymes, kinases and phosphatases, tightly control all phosphorylation events. Perturbation in the activity of any member of these classes of enzymes has been linked to numerous diseases including cancer, metabolic disorders, immune disorders and neurological disorders. Therefore, there is a great interest among the scientific community to develop methods to selectively modulate the activity of kinases and phosphatases not only for therapeutic purposes but also to understand the fundamental role of these enzymes in signaling events. The more than 500 kinases encoded in the human genome share a common catalytic fold and most inhibitors target the ATP binding site. Therefore selective targeting of a single kinase by an inhibitor at the highly conserved ATP binding site is one of the main concerns for designing probes or drugs. Our group has taken advantage of the potency and possible selectivity imparted by bivalent inhibitors and developed an in vitro selection approach to discover bivalent ligands. The strategy involves the use of an ATP-competitive small molecule warhead and a library of cyclic peptides displayed on phage that interact with the kinase of interest in a dynamic selection. The selection for a kinase binding peptide is carried out until consensus peptides are found and bivalent ligands are constructed by linking the selected cyclic peptide with the small molecule warhead through a synthetic linker. Using this approach a potent and selective bivalent inhibitor was found for PKA, a serine/threonine kinase. To interrogate the generality of this approach, a kinase closely related to PKA (PRKX) was used for which a very potent and selective bivalent ligand was found. The same selection strategy was further extended to the two kinases Lyn and Brk, which belong to the tyrosine kinase family. Though peptides were isolated that bound the desired kinase, potent bivalent inhibitors were not discovered. More generally, these experiments in sum are building a library of information regarding how to best design selections of potent and selective bivalent inhibitors. We further explored modulation of the activity of kinases and phosphatases by employing a ligand-gated split-protein approach. The small molecule gated spatial and temporal control of these enzymes should allow the study of signaling events in a controlled manner. The strategy employed consists in the identification of possible fragmentation sites within the catalytic domain of kinases and phosphatases by a sequence dissimilarity approach. Loop insertion mutants at the selected sites were tested for catalytic activity. Successful insertion mutants were further split into two catalytically inactive fragments, which were appended to two conditionally interacting protein domains. Upon addition of a small molecule, the two conditionally interacting domains reassemble the catalytic domain of the enzyme and restore catalytic activity. Using this approach we were able to modulate the activity of the tyrosine kinases Lyn, Fak and Src and the AGC kinase PKA. We also extended the approach to gate the activity of tyrosine phosphatases PTP1B, SHP1 and PTPH1. Finally, these ligand-gated split-kinases and phosphatases were validated in-cellulo. Thus, this work resulted in a new method for designing split-proteins and provided a palette of kinases and phosphatases that can be turned-on by small molecules. In total, these efforts describe two alternative routes that can be used to modulate phosphorylation events in a selective and controlled manner.

The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 128 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 118-127).

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2007. / Title from PDF t.p. (viewed on Apr. 16, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2005. / Title from title screen. Includes bibliographical references.

Thesis supervisor: Stefan Strack. Includes bibliographic references (p. 87-100).

Thesis (M.S.)--Brock University, 2009. / Includes bibliographical references (leaves 69-85).

Thesis (doctoral)--Lund University, 1995.

Thesis (doctoral)--Lund University, 1995.

Thesis (Ph. D.)--Illinois State University, 2005. / Title from title page screen, viewed September 26, 2006. Dissertation Committee: Anthony J. Otsuka (chair), Radheshyam Jayaswal, Kevin A. Edwards, David L. Williams, Hou Tak Cheung. Includes bibliographical references (leaves 110-124) and abstract. Also available in print.